CN100405065C - Nano colloid gold reagent strip for immunochromatography detection of bladder carcinoma and detection method thereof - Google Patents
Nano colloid gold reagent strip for immunochromatography detection of bladder carcinoma and detection method thereof Download PDFInfo
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- CN100405065C CN100405065C CNB2005101047966A CN200510104796A CN100405065C CN 100405065 C CN100405065 C CN 100405065C CN B2005101047966 A CNB2005101047966 A CN B2005101047966A CN 200510104796 A CN200510104796 A CN 200510104796A CN 100405065 C CN100405065 C CN 100405065C
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Abstract
The present invention discloses a nanometer colloidal gold immunochromatography detecting reagent for bladder cancer and a detecting method thereof. The present invention relates to a urine detecting apparatus, particularly to o a reagent for detecting bladder cancer by utilizing an immunochromatographic method and nanometer colloidal gold technology and a detecting method thereof. The present invention provides an immunochromatography detecting reagent strip for bladder cancer and a detecting method thereof. A urine absorbing glass fiber layer, a polyester film layer, a glass fiber layer on which gold labeled anti-CK19 antibodies or Ks19.2 antibodies are fixed, a cellulose nitrate film layer and a water absorbing filter paper layer are orderly arranged on a bottom board from left to right. The urine absorbing glass fiber layer, the polyester film layer, the glass fiber layer on which gold labeled anti-CK19 antibodies, the cellulose nitrate film layer and the water absorbing filter paper layer are orderly connected and pasted in an overlapping mode on the bottom board to be assembled into the immunochromatography detecting reagent strip for bladder cancer. A T test region and a C quality control region are arranged on the cellulose nitrate film layer, wherein the test region is coated with gold labeled anti-CK19 antibodies or Ks19.2 antibodies, and the quality control region is coated with sheep anti-mouse antibodies.
Description
Technical field
The present invention relates to a kind of urine detection utensil, especially relate to a kind of reagent strip and detection method thereof of utilizing immunochromatographic method (immuno-chromatography) and nano colloid gold technology for detection carcinoma of urinary bladder.
Background technology
Immunochromatographic method is a kind of quick diagnosis technology of rising in recent years, its principle is a certain district band that special antibody is fixed in nitrocellulose filter earlier, after this dry nitrocellulose filter one end immerses sample, because capillarity, sample will move forward along this film.When moving to when being fixed with antibody regional, corresponding antigen promptly combines with this antibody generation specificity in the sample.If can make this zone show certain color with immune colloid gold or immuno-enzymatic dyeing, thereby realize specific immunodiagnosis.Its advantage be simplify the operation step, accelerate detection speed, specificity is good, highly sensitive, is applicable to generaI investigation and screening and scene (bedside) diagnosis of clinical labororatory.
The colloidal gold chromatographic technology is a kind of new technology (the Beggs M that grows up the eighties in 20th century, Novotany M, Sampedro S.A selfperforming chromatographic immunoassay for the qualitativedetermination of human chorionic gonadotrophin (HCG) in ureine and serum[J] .ClinChem 1990,36:1084-1085.) since it with since immunological method combines, development is rapidly.It reacts needed whole or most of raw material and all is incorporated in the reagent.After testing sample (urine, serum or organic solvent extraction liquid) joins on the sample film, because the capillarity of miillpore filter, antigen-antibody reaction is carried out on immobilon-p fast, reaction only needs several minutes usually, its test result is brought judgement with macroscopic colour developing bar, and its chromogenic reaction is to assemble at conversion zone and form owing to the coloured particle of institute's mark.Gold mark monoclonal antibody fast diagnosis reagent have high specificity, susceptibility height, simple and easy fast, reagent is easily preserved and with low cost and do not need advantage such as specific installation.
Tumor of bladder is higher than industrial underdeveloped countries at the incidence of disease of industrialized country, and the incidence of disease in the city is higher than the rural area, the destruction of environmental pollution and ecologic environment, and the contact of carcinogen is the main cause that the carcinoma of urinary bladder incidence of disease increases.
Diagnosis of bladder cancer is a goldstandard with exfoliative cell examination of urine and cystoscope biopsy.But the urine sample cytolgical examination, influence factor is more, and positive rate is low, for I, II phase patient positive rate less than 50%.
Clinical early diagnosis refers to that tumour comes across after the morphological change more, uses CT usually, B ultrasonic carries out early diagnosis, but tumour a hour leakiness examine, and the character of tumour is difficult for judging.Cystoscope is than CT, B ultrasonic diagnosis height, but carcinoma in situ is difficult to diagnosis.Under the situation of not doing pathological section, cystoscope is thought reliable diagnostic method, but this method is the traumatic inspection of invasive, bring misery to patient, and charge is high, the patient is difficult for accepting, and be only limited to the diagnosis of observability tumour, and the chance that increases urinary system infection contamination is arranged.
Applied Electrochemistry luminescent immunoassay technology for detection urine CYFRA211 has susceptibility and negative predictive value preferably to diagnosis of bladder cancer, and quick and AT advantage is arranged, and can be used as the auxiliary diagnosis index and the monitoring after operation index of carcinoma of urinary bladder.But cost is too high, can not be used for the bedside inspection, and needs valuable electrochemiluminescence instrument, the medical condition of incompatibility basic hospital and backward areas.
(cytokeratin CKs) is epithelial structural proteins to cytokeratin, has found 20 kinds of different cytokeratins at present.Cytokeratin 19 fragments (cytokeratin 19fragment, cytokeratin 211) be one of cytokeratin composition, molecular weight 43kD, it is the soluble acid polypeptide, CK19 content is very low under the normal condition, and when cell carcinogenesis, content raises, because of cell permeability changes or dissolving are released, make that concentration raises in tissue fluid, the body fluid.Urine CK19 level is meaningful to the monitoring of diagnosis of bladder cancer and postoperative recurrence.During the carcinoma of urinary bladder epithelial cell canceration, owing to tumor cell proliferation, come off, dissolve, the cytokeratin composition is overflowed and degraded in cell, makes CK19 level rising in the urine.Research (Zhang Zhongying is arranged, woods is always cherished the memory of, and yellow pine is clean, etc. the meaning [J] of keratin 19 fragments in the electrochemiluminescence technology for detection urine. Chinese laboratory medicine magazine, 2003,26 (2): 104-106.) show that urine CK19 has higher susceptibility (93.3%) and specificity (82.7%) to carcinoma of urinary bladder.
Summary of the invention
The object of the present invention is to provide the application of the anti-CK19 antibody of gold mark (antibody of the anti-CK19 fragment of colloid gold label, English Monoclonal Antibody to Cytokeratin 19 by name) in a kind of reagent strip for immunochromatographydetection detection of bladder carcinoma of preparation.
Another object of the present invention is to provide a kind of reagent strip for immunochromatographydetection detection of bladder carcinoma and detection method thereof.
The gold anti-CK19 antibody of mark (Monoclonal Antibody to Cytokeratin 19) can be used for preparing a kind of reagent strip for immunochromatographydetection detection of bladder carcinoma, and is used for the detection of carcinoma of urinary bladder.
Reagent strip for immunochromatographydetection detection of bladder carcinoma of the present invention is provided with base plate, on base plate, from left to right be provided with successively and inhale the urine glass layer, the polyester rete, be fixed with gold mark anti-CK19 antibody (Monoclonal Antibody to Cytokeratin19) or Ks19.2 antibody (Clone Determination Ks 19.2 (Z105.6), also published as BM 19.21, MAK 19.21) glass layer, cellulose nitrate rete (NC film) and absorbent filter layer are inhaled the urine glass layer, the polyester rete, be fixed with the glass layer of the anti-CK19 antibody of gold mark, cellulose nitrate rete (NC film) and absorbent filter layer be overlapping successively to link to each other to stick on and assembles on the base plate.
T test section and C Quality Control district are arranged on the cellulose nitrate rete.The test section is that bag is by gold mark anti-CK19 antibody (Monoclonal Antibody to Cytokeratin 19) or Ks19.2 antibody (Clone Determination Ks19.2 (Z105.6), also published as BM 19.21, MAK 19.21), the Quality Control district is that bag is by sheep anti-mouse antibody.Wherein inhaling the urine glass layer is sample application zone.
The cellulose nitrate rete is of a size of: long 2～4.5cm, and wide 2.5～4.5mm is preferably long 2.5cm, wide 3.5mm, base plate can be selected cardboard, plastic plate etc. for use.
The concrete steps of carcinoma of urinary bladder immunochromatography detection method of the present invention are as follows:
1) get 30～150 μ L, best 50 μ L sample to be checked is added in the sample application zone of reagent strip.
2), preferably observe and log in the 5min at 2～30min.
The present invention adopts the CK19 fragment in the colloidal gold immunochromatographimethod technology double antibody sandwich method principle test sample, detects whether there is the CK19 antigene fragment from urine, so that as the auxiliary reference parameter of diagnosing bladder cancer.Drip urine in sample application zone, because of the chromatography effect, flow direction absorbent filter direction, when flowing to golden label place, golden labeling antibody dissolving, and combine with CK19 fragment antigen in the urine and to form compound.When gold is marked anti-CK19 antibody-CK19 fragment antigen compound and is continued reach, combine with coated antibody at p-wire, form " gold mark anti-CK19 antibody-antigen-Bao is by Ks19.2 antibody " compound and condense colour developing, form the anti-CK19 antibody-Bao of gold mark at the nature controlling line place and developed the color by the sheep anti-mouse antibody compound, not developing the color as nature controlling line shows that then this reagent is invalid.
The present invention compares with the colloidal gold diagnosis kit of the uroscopy carcinoma of urinary bladder that the U.S. adopts, both principle differences, and BTAstat detects people's complement factor H associated protein (hCFHrp), and its 26S Proteasome Structure and Function is similar to people's complement factor H (hCFH).HCFH suppresses alternative pathway of complement and foreign cell cracking.Similar to hCFH, the reaction of BTA blocking-up complement cascade makes cancer cell escape the supervision of host immune system.And the present invention adopts the CK19 fragment in the colloidal gold immunochromatographimethod technology double antibody sandwich method principle test sample, detects whether to have the CK19 antigene fragment from urine, so that as the auxiliary reference parameter of diagnosing bladder cancer.Drip urine in sample application zone, because of the chromatography effect, flow direction absorbent filter direction, when flowing to golden label place, golden labeling antibody dissolving, and combine with CK19 fragment antigen in the urine and to form compound.When gold is marked anti-CK19 fragment antibody-CK19 fragment antigen compound and is continued reach, combine with coated antibody at p-wire, form " gold mark anti-CK19 antibody-antigen-Bao is by Ks19.2 antibody " compound and condense colour developing, form the anti-CK19 antibody-Bao of gold mark at the nature controlling line place and developed the color by the sheep anti-mouse antibody compound, not developing the color as nature controlling line shows that then this reagent is invalid.
Compare with other detection methods of carcinoma of urinary bladder, the present invention has following outstanding advantage:
1) good, the specificity of susceptibility and accuracy height and non-invasive can detect the CK19 fragment in the urine, and the sample consumption only needs 50 μ L, can detect nanogram level CK19 fragment, are applicable to that multiple occasion detects.Its sensitivity can reach 5～10ng/mL, and bladder transitional cell cancer patient's recall rate can reach more than 85%.
2) detection speed is fast, cost is low, can in 5min, go out the result, simple and efficient to handle, be applicable to that the epidemiology survey auxiliary detection of tumor of bladder or dynamic monitoring and the clinician after the treatment screen cystoscopy adaptation population (promptly finding out highly suspicious bladder cancer patients with this diagnostic kit).
Description of drawings
Fig. 1 is the structural representation of reagent strip for immunochromatographydetection detection of bladder carcinoma of the present invention.
Embodiment
Following examples will the present invention is further illustrated in conjunction with the accompanying drawings.
Referring to Fig. 1, reagent strip for immunochromatographydetection detection of bladder carcinoma is provided with cardboard base plate 1, on base plate 1, from left to right be provided with suction urine glass layer 2, polyester rete 3 successively, be fixed with the anti-CK19 antibody of gold mark (Monoclonal Antibody toCytokeratin 19, glass layer 4 clone:A53-B/A2), cellulose nitrate rete 5 (NC film) and absorbent filter layer 6.Each layer quilt be staggered successively to be bonded in and to assemble on the cardboard base plate 1.T test section 7 and C Quality Control district 8 are arranged on cellulose nitrate rete 5.Test section 7 is the district's bands that indicate Ks19.2 antibody (MAK 19.21 for Clone Determination Ks19.2 (Z105.6), also published as BM 19.21), and Quality Control district 8 indicates district's band that anti-mouse two resists.Wherein inhale urine glass layer 2 (district) as sample application zone.Cellulose nitrate rete 5 is of a size of: long 2.5cm, wide 3mm, base plate also can select plastic plate etc. for use.Carrying out the carcinoma of urinary bladder immunochromatography when detecting, getting the sample application zone that 50 μ L sample to be checked is added in reagent strip earlier and promptly inhale on the urine glass layer 2, in 5min, observing and log.
The judgement of testing result is as follows:
Positive findings: on the p-wire of reagent strip and nature controlling line position, an aubergine band respectively occurs.
Negative findings: only occur an aubergine band on the nature controlling line position of reagent strip.
Null result: on the p-wire of reagent strip and nature controlling line position, the aubergine band all do not occur.
When detecting, should note following item:
1) test sample is in room temperature (about 20 ℃) balance and detect under this condition.The requirement test sample is a fresh sample.Before making a collection of specimens, patient should control the water yield and take in, and leaves and takes urina sanguinis, midstream urine, and is in time centrifugal, and methods such as timely mensuration are to guarantee result's reliability.
2) when antibody content is high in the sample, the nature controlling line band weakens to some extent, and this result still is considered as effective result (if need further conclusive evidence, can will remake test after an amount of dilution of sample).
3) this reagent strip only donor detect primary dcreening operation outward, as confirming reagent, positive findings must not carry out the confirmatory test conclusive evidence.
4) observations is invalid behind the 30min.
5) 4～30 ℃ of lucifuge kept dry.
Referring to Fig. 1, similar to Example 1, its difference is to be fixed with on the glass layer 4 Ks19.2 antibody (CloneDetermination Ks 19.2 (Z105.6), also published as BM 19.21, MAK 19.21), the test section on cellulose nitrate rete 5 indicates the anti-CK19 antibody of gold mark (Monoclonal Antibody to Cytokeratin 19, district's band clone:A53-B/A2).Cellulose nitrate rete 5 is of a size of: long 2cm, wide 3.5mm.
Carrying out the carcinoma of urinary bladder immunochromatography when detecting, getting the sample application zone that 100 μ L sample to be checked is added in reagent strip earlier and promptly inhale on the urine glass layer 2, in 2min, observing and log.The judgement of testing result is identical with embodiment 1.
Referring to Fig. 1, similar to Example 2, its difference is that cellulose nitrate rete 5 is of a size of: long 4.5cm, wide 4.5mm.
Carrying out the carcinoma of urinary bladder immunochromatography when detecting, getting the sample application zone that 30 μ L sample to be checked is added in reagent strip earlier and promptly inhale on the urine glass layer 2, in 15min, observing and log.The judgement of testing result is identical with embodiment 1.
Referring to Fig. 1, similar to Example 1, its difference is that cellulose nitrate rete 5 is of a size of: long 3cm, wide 2.5mm.
Carrying out the carcinoma of urinary bladder immunochromatography when detecting, getting the sample application zone that 150 μ L sample to be checked is added in reagent strip earlier and promptly inhale on the urine glass layer 2, in 30min, observing and log.The judgement of testing result is identical with embodiment 1.
Patient Xu * *, the man 32 years old, makes a definite diagnosis carcinoma of urinary bladder in the court.Its urine sample electricity consumption chemiluminescence immunoassay technology and detectable bar of the present invention detect respectively.
1 collection of specimens: collect the urine sample, lighted fasting the evening before yesterday 9 that makes a collection of specimens and prohibit water, stay the 1st about 5mL of midstream urine in early morning, place dedicated pipe, put upside down mixing, censorship in the 3h in 4 ℃ of preservations.
2 sample disposal: with specimen centrifuge, the centrifugal 5min of 1000～3500r/min, preferably the centrifugal 5min of 1500r/min gets machine testing on the supernatant (or-75 ℃ of preservations).
3 methods:
1) adopts electrochemiluminescence immuno analytical method (ECLIA).Electrochemiluminescence immunity analysis instrument (Elecsys1010 type, Roche company product), the CYFRA21-1ECLIA kit is provided by Roche company, and strictness is operated by operational manual.
2) get the sample application zone that 50 μ L urine sample to be checked is added in reagent strip of the present invention and promptly inhale on the urine glass layer, in 5min, observe and log.
The result: the CK19 (CYFRA21-1) that electrochemiluminescence immuno analytical method (ECLIA) is measured is 5.16 μ g/L; The reagent strip display result is positive, promptly an aubergine band respectively occurs on the p-wire of reagent strip and nature controlling line position.
Claims (4)
1. reagent strip for immunochromatographydetection detection of bladder carcinoma, it is characterized in that being provided with base plate, on base plate, from left to right be provided with successively and inhale urine glass layer, polyester rete, glass layer, cellulose nitrate rete and absorbent filter layer, inhale urine glass layer, polyester rete, glass layer, cellulose nitrate rete and absorbent filter layer overlapping successively link to each other to stick on assemble on the base plate;
On glass layer, be fixed with gold anti-CK19 antibody of mark or Ks19.2 antibody;
T test section and C Quality Control district are arranged on the cellulose nitrate rete, and the test section bag is by gold anti-CK19 antibody of mark or Ks19.2 antibody, and wrap by sheep anti-mouse antibody in the Quality Control district;
Described suction urine glass layer is a sample application zone.
2. reagent strip for immunochromatographydetection detection of bladder carcinoma as claimed in claim 1 is characterized in that the cellulose nitrate rete is of a size of long 2～4.5cm, wide 2.5～4.5mm.
3. reagent strip for immunochromatographydetection detection of bladder carcinoma as claimed in claim 2 is characterized in that the cellulose nitrate rete is of a size of long 2.5cm, wide 3.5mm.
4. reagent strip for immunochromatographydetection detection of bladder carcinoma as claimed in claim 1 is characterized in that base plate is selected from cardboard or plastic plate.
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| CNB2005101047966A CN100405065C (en) | 2005-12-27 | 2005-12-27 | Nano colloid gold reagent strip for immunochromatography detection of bladder carcinoma and detection method thereof |
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| CNB2005101047966A CN100405065C (en) | 2005-12-27 | 2005-12-27 | Nano colloid gold reagent strip for immunochromatography detection of bladder carcinoma and detection method thereof |
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| CN100405065C true CN100405065C (en) | 2008-07-23 |
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| CN104280547A (en) * | 2013-08-12 | 2015-01-14 | 云南师范大学 | Application method of CK15 keratin and gene |
| CN105572383A (en) * | 2016-01-12 | 2016-05-11 | 深圳大学 | Colloidal gold chromatographic test strip for detecting cytokeratin 19 (CK19) and preparation method thereof |
Citations (5)
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| US20030044977A1 (en) * | 2001-08-10 | 2003-03-06 | Norio Sakuragawa | Human stem cells originated from human amniotic mesenchymal cell layer |
| CN1458527A (en) * | 2002-08-21 | 2003-11-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chromatographic paper strip and method for quick detecting pathogen and toxin in food |
| CN1553193A (en) * | 2003-06-06 | 2004-12-08 | 卢氏实验公司 | Counter colloidal gold immune chromographic reagent strips and their use |
| WO2005118875A2 (en) * | 2004-06-04 | 2005-12-15 | Veridex, Llc | Diagnosing or predicting the course of breast cancer |
| CN2862034Y (en) * | 2005-12-27 | 2007-01-24 | 厦门大学附属中山医院 | Colloidal gold bladder cancer checking reagent strip |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030044977A1 (en) * | 2001-08-10 | 2003-03-06 | Norio Sakuragawa | Human stem cells originated from human amniotic mesenchymal cell layer |
| CN1458527A (en) * | 2002-08-21 | 2003-11-26 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chromatographic paper strip and method for quick detecting pathogen and toxin in food |
| CN1553193A (en) * | 2003-06-06 | 2004-12-08 | 卢氏实验公司 | Counter colloidal gold immune chromographic reagent strips and their use |
| WO2005118875A2 (en) * | 2004-06-04 | 2005-12-15 | Veridex, Llc | Diagnosing or predicting the course of breast cancer |
| CN2862034Y (en) * | 2005-12-27 | 2007-01-24 | 厦门大学附属中山医院 | Colloidal gold bladder cancer checking reagent strip |
Non-Patent Citations (2)
| Title |
|---|
| 电化学发光免疫分析法检测尿细胞角蛋白19及其在膀胱癌诊断中的应用. 叶辉铭等.检验医学，第20卷第6期. 2005 |
| 电化学发光免疫分析法检测尿细胞角蛋白19及其在膀胱癌诊断中的应用. 叶辉铭等.检验医学，第20卷第6期. 2005 * |
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