CN106546570A - A kind of detection method of ABCG2 antibody - Google Patents
A kind of detection method of ABCG2 antibody Download PDFInfo
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- CN106546570A CN106546570A CN201610954271.XA CN201610954271A CN106546570A CN 106546570 A CN106546570 A CN 106546570A CN 201610954271 A CN201610954271 A CN 201610954271A CN 106546570 A CN106546570 A CN 106546570A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a kind of ABCG2 antibody detection methods, belong to the method for determining internal blood characteristics.Negative control sera of the kit of the present invention built with detection, positive control serum, concentrated phosphoric acid buffer solution, rabbit anteserum confining liquid, biotinylation rabbit-anti people's ABCG2 antibody, the reagents such as fluorescein-labeled Streptavidin and bio-sheet material, bio-sheet material are made by taking rat kidney proximal tubule, are coated in the reaction zone of slide glass；Assay method is：Add test serum in reaction zone, Jing after incubation, rinsing, drying, add biotinylation rabbit-anti people's ABCG2 antibody, Jing after incubation, rinsing, drying, add fluorescein-labeled Streptavidin, fluorescein-labeled Streptavidin biotin fluorescent composition is formed, and produces yellow-green fluorescence, the inventive method is above existing method in indexs such as sensitivity, specificity.
Description
Technical field
The present invention relates to measure the detection method of internal blood characteristics, the specifically detection method of ABCG2 antibody.
Background technology
Abc transport albumen (ATP-binding cassette transporters, ABCG2) is using the energy of hydrolysising ATP
Transmembrane transport is carried out to sugar, amino acid, metal ion, polypeptide, protein, uric acid, products of cellular metabolism and medicine, its gene position
In 4q22～23,655 amino acid residues are encoded, zoopery confirms that the serum uric acid level of ABCG2 mutant mices increases, in people
The mutation of class ABCG2 is considered as the major reason of gout and hyperuricemia, particularly familial gout and young antihyperuricemic
The main cause of disease, such as change of ABCG2 functions, chordapsus etc., cause the uric acid of enteral road excretion to reduce, then so that kidney
Dirty uric acid load increase, causes blood uric acid to raise.
Uric Acid total amount is 0.9～1.6 gram, about updates 60% daily, produces 750 milligrams daily, and acid-base value is 5.75,
Body fluid meta-alkalescence, in human body, uric acid daily capacity and excretion are approximately equivalent, make one internal uric acid and keep balance, uric acid excretion
Approach is then 1/3 to be discharged by enteron aisle, and 2/3 from RE, the circulation excretion of small part Jing liver and gall.
ABCG2 gene high expressions encode a kind of adenosine triphosphate dependent Ser in enterocyte, renal proximal tubules teleblem
Uric acid secretes molecule, and the major function of ABCG2 genes is that, in enteron aisle, renal secretion uric acid transporter body, ABCG2 genes are in the outer group of kidney
Knit (include liver) high expression.The genetic mutation most species of ABCG2, the impact to serum uric acid level are maximum, in addition to kidney,
The outer adjuster of kidney of ABCG2 also uric acid excretions, its in addition to being expressed on kidney proximal tubule BBM, also big scale
Up in the top film and liver cell of intestinal epithelial cell, it is responsible for metabolism of the uric acid outside kidney, by the gene to ABCG2
Sequence analysis finds that what it had overshoot section uric acid excretion is the ABCG2 albumen of enteron aisle and kidney, equal in kidney and enteron aisle ABCG2
Positioned at the tube chamber side form of epithelial cell, when there is chordapsus, intestinal mucosa ABCG2 protein conformations change, function limitation,
Intestinal transit uric acid function reduction, renal cells ABCG2 albumen are unaffected, cause blood uric acid concentration to raise, when
When sb.'s illness took a favorable turn, ABCG2 protein functions recover, uric acid transporter functional rehabilitation, and uric acid in blood declines.
Hyperuricemia is caused by purine metabolic disturbance, kidney trouble, enteritis etc., is regulated and controled by ABCG2 protein functions, it
Function also reflect enteron aisle, renal function drain uric acid function.
When occurring ABCG2 protein antibodies in human body, there is the change of transport function in internal ABCG2 albumen, and then occurs
Uric acid excretion disorder, causes internal blood uric acid to raise, therefore, carry out the detection of ABCG2 protein antibodies in human body to antihyperuricemic
The treatment of disease is significant.
The content of the invention
There is the defect failed to pinpoint a disease in diagnosis to solve ABCG2 antibody detection methods in known technology in the present invention, and provide a kind of accurate
Really property is high, the detection method of more sensitive ABCG2 antibody.
The technical solution used in the present invention is：
A kind of ABCG2 antibody detection methods, it is characterised in that take rat kidney proximal tubule and make bio-sheet material as antigen,
It is coated in the reaction zone of slide；Add dilute serum to be measured in reaction zone, Jing after incubation, rinsing, drying, add biotin
Change rabbit-anti people's ABCG2 antibody, add fluorescein-labeled Streptavidin, form fluorescein-labeled Streptavidin-life
Thing element fluorescent composition, and produce yellow-green fluorescence.
Fluorescence detecting system is introduced using by biotin-Streptavidin system, sensitiveness can be significantly improved and the back of the body is reduced
Scape, improves sensitiveness using the enlarge-effect and Streptavidin-multienzyme complex of the system, and using the advantage of fluorescence, commonly uses
Fluorescein remain fluorescein isothiocynate (FITC), FITC is excited in 495nm, 525nm launch fluorescence, in green it is glimmering
Streptavidin-biotin system is introduced fluorescing system by light, can be significantly improved sensitiveness and be reduced background.
One. box body
The kit of the present invention is hexahedral shape, is made up of with cassette bottom lid, is characterised by cassette bottom near long side
Any a line be provided with place reagent bottle bottle stand；Bottle stand is provided with multiple holes for placing reagent bottle；The centre of cassette bottom sets
Have one cassette bottom is divided into placement reagent and the two-part catch of biological slide glass is placed, including quadrangle box (1) and with its side
Side bends the lid (2) of disjunctor, it is characterised in that：Reagent rack (3) is set in box body (1), in reagent rack, there are 6 same apertures
Jack (4), is placed with rabbit anteserum confining liquid (9), biotinylation rabbit-anti people's ABCG2 antibody (10), fluorescein isothiocynate in jack
Coupling Streptavidin (11), negative control sera (12), positive control serum (13), mountant (14), the centre of cassette bottom sets
There is one cassette bottom is divided into placement buffering agents (8) and the two-part papery catch (6) of bio-sheet material (5) is placed, it is biological to carry
Plastics package (7) outside piece (5).
Description of the drawings
Fig. 1 is the schematic diagram of kit of the present invention；
Fig. 2 is the schematic diagram of slide glass of the present invention.
1. 2. lid of box body
3. 4. jack of reagent rack
5. 6. papery catch of bio-sheet material
7. 8. buffer solution of bio-sheet material plastics package
9. 10. biotinylation rabbit-anti people's ABCG2 antibody of rabbit anteserum confining liquid
11. fluoresceinisothiocyanate Streptavidin, 12. negative control sera
13. positive control serum, 14. mountant
Wherein on bio-sheet material (5), 5a, 5b, 5c, 5d, 5e are test reaction area.
Two. the article that kit is equipped with
1 bottle negative control sera, positive control serum, mountant, rabbit are placed with the bottle stand 3 of kit of the present invention respectively
Serum block, biotinylation rabbit-anti people's ABCG2 antibody, fluoresceinisothiocyanate Streptavidin, cassette bottom left half are put
Phosphate (containing 10.2 grams of the buffered phosphate) buffer solution of two bottles of 50ml concentrations is equipped with, right half is placed with 8 with packaging bag
Bio-sheet material.
3rd, the making of biological slide
The material of thin slice of the present invention 5 is polystyrene, and its size is similar to general slide glass.Which simultaneously scribbles polylysine painting
Layer, arranges five square reaction zone 5a, 5b, 5c, 5d, 5e on slide glass.Reaction zone is cated one towards inside slide glass
Depression, forms the groove of 25 μ l volumes, and the groove part of each reaction zone is coated with the life of a rat kidney proximal tubule frozen section
Thing thin slice, is surrounded by one layer of lighttight packaging bag outside slide glass, 2-8 DEG C of preservation in refrigerator.
Specific embodiment
Embodiment 1
1. detection method
After taking bio-sheet material placement room temperature, 0.01mol/L PBS1 are added dropwise:The normal rabbit serum confining liquid of 10 dilutions, room temperature
20min, 0.01mol/L PBS washes 2min 3 times.Add 1：The serum of 100PBS dilutions, 20～37 DEG C 1～2h or 4 DEG C is overnight,
0.01mol/L PBS wash 2min 3 times；0.01mol/L PBS1 are added dropwise:Rabbit-anti people's biotinylation ABCG2 antibody of 100 dilutions,
20～37 DEG C of 1～2h, 0.01mol/L PBS wash 2min 3 times；0.01mol/L PBS1 are added dropwise:The isothiocyanic acid of 100 dilutions is glimmering
Light element is coupled Streptavidin, and 20～37 DEG C of 1～2h, 0.01mol/L PBS wash 2min 4 times；Mountant mounting, fluorescence microscopy
Observe under 10 X of mirror, 40 times of visuals field；FITC is excited in 490～495nm, launches fluorescence in 520～530nm, such as positive in yellowish green
Color.
2. ELISA (commercially available)
2.1. ELISA kit is taken out from refrigerator, is put to room temperature, be determined according to sample quantity to be checked
ELISA Plate quantity.
2.2. sample-adding：Set respectively blank well (blank control wells are not added with sample and enzyme marking reagent, and remaining each step operation is identical),
Testing sample hole.First add 40 μ l of sample diluting liquid in testing sample hole on enzyme mark coating plate, then add 10 μ l of testing sample again
(the final dilution factor of sample is 5 times).Sample is added on ELISA Plate bottom hole portion by sample-adding, is not touched hole wall as far as possible, is gently rocked mixing.
2.3. incubate：With shrouding film shrouding, rearmounted 37 DEG C incubate 30 minutes.
2.4. match somebody with somebody liquid：By 20 times of concentrated cleaning solutions with standby after the dilution of 20 times of distilled water.
2.5. washing：Carefully take shrouding film off, discard liquid, dry, cleaning solution is filled it up with per hole, discard after standing 30 seconds,
So it is repeated 5 times, pats dry.
2.6. it is enzyme-added：50 μ l of enzyme marking reagent are added per hole, except blank well.
2.7. incubate：With shrouding film shrouding, rearmounted 37 DEG C incubate 30 minutes.
2.8. washing：Carefully take shrouding film off, discard liquid, dry, cleaning solution is filled it up with per hole, discard after standing 30 seconds,
So it is repeated 5 times, pats dry.
2.9. colour developing：50 μ l of developer A are initially charged per hole, 50 μ l of developer B are added, gently concussion is mixed, 37 DEG C
Lucifuge develops the color 15 minutes.
2.10. terminate：Add 50 μ l of terminate liquid, terminating reaction per hole (now blue standing turns yellow).
2.11. determine：With blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance (OD values) in each hole, determines Ying Jia
Carry out within 15 minutes after terminate liquid.
3. for a better understanding of the present invention, below by the result drawn with two kinds of detection methods further illustrating this
Good effect of the invention in terms of detection ABCG2 antibody：
In January, 2015 in January, 2016 is at our hospital's Patients with Hyperuricemia 58, the male sex 30, women 28, age 40
～60 years old, average 48.5 years old, the course of disease 4 months to 5.7 years, average 3.6 years, control group was our hospital medical examiner, age and sex
Match with patient's group.
ABCG2 antibody detection methods：1. detection method；2. ELISA.Be respectively adopted this two
The method of kind detects 58 Patients with Hyperuricemia and control group, and two methods are carried out statistical analysis.
The two methods detection high lithemia group of table 1, control group statistics comparison sheet.
Jing Chi-square Tests, relatively have significant difference, Jing t inspection P in two methods of high lithemia group<0.05；In control group
Two methods compare that there was no significant difference, P>0.05.
2 two methods testing result of table compares (%)
The method of testing of the present invention can be seen in sensitivity by table 1, table 2, specificity, positive predictive value are above enzyme-linked exempting from
Epidemic disease adsorption method, can be used as the early diagnosis of patient with gout ABCG2 antibody.
Claims (4)
1. a kind of detection method of ABCG2 antibody, it is characterised in that take after bio-sheet material places room temperature, 0.01mol/L is added dropwise
PBS1:The rabbit anteserum confining liquids of 10 dilutions, room temperature 20min, 0.01mol/L PBS wash 2min 3 times, add 1：100PBS dilutes
Serum, 20～37 DEG C 1～2h or 4 DEG C is overnight, and 0.01mol/L PBS wash 2min 3 times；0.01mol/L PBS1 are added dropwise:100
Rabbit-anti people's biotinylation ABCG2 antibody of dilution, 20～37 DEG C of 1～2h, 0.01mol/L PBS wash 2min 3 times；It is added dropwise
0.01mol/L PBS1:The fluoresceinisothiocyanate Streptavidin of 100 dilutions, 20～37 DEG C of 1～2h, 0.01mol/L
PBS washes 2min 4 times；Mountant mounting, observes under 10 X of fluorescence microscope, 40 times of visuals field；FITC is excited in 490～495nm,
520～530nm launches fluorescence, such as positive in yellow green.
2. the detection method of a kind of ABCG2 antibody according to claim 1, it is characterised in that mentioned reagent box includes feminine gender
Control serum, positive control serum, concentrated phosphoric acid buffer solution, rabbit anteserum confining liquid, biotinylation rabbit-anti people's ABCG2 antibody, it is different
Thiocyanic acid fluorescein is coupled Streptavidin.
3. the detection method of a kind of ABCG2 antibody according to claim 1, it is characterised in that have 5 reaction zones on slide glass,
Each reaction zone is coated with a rat kidney proximal tubule and makes bio-sheet material.
4. the detection method of a kind of ABCG2 antibody according to claim 1, it is characterised in that rabbit-anti people's ABCG2 antibody is
Biotinylated antibody, fluorescent marker are fluoresceinisothiocyanate Streptavidin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610954271.XA CN106546570A (en) | 2016-11-03 | 2016-11-03 | A kind of detection method of ABCG2 antibody |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610954271.XA CN106546570A (en) | 2016-11-03 | 2016-11-03 | A kind of detection method of ABCG2 antibody |
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| CN106546570A true CN106546570A (en) | 2017-03-29 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110806478A (en) * | 2019-11-24 | 2020-02-18 | 天津市宝坻区人民医院 | Clostridium difficile detection kit |
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| CN201828517U (en) * | 2010-09-25 | 2011-05-11 | 天津市宝坻区人民医院 | Kit for detecting hand-foot-and-mouth disease by adopting biotin fluorescence method |
| CN201892679U (en) * | 2010-09-25 | 2011-07-06 | 天津市宝坻区人民医院 | Influenza A H1N1 immunofluorescence detection reagent box |
| CN102532298A (en) * | 2012-03-01 | 2012-07-04 | 刘林林 | ABCC3 antigen polypeptide specially binding with autoantibody and application |
| CN204188622U (en) * | 2014-10-09 | 2015-03-04 | 天津市宝坻区人民医院 | Antikeratin antibody detection kit |
| CN105759029A (en) * | 2016-02-24 | 2016-07-13 | 天津市宝坻区人民医院 | Detection kit for measles virus and application method of detection kit |
| CN106018805A (en) * | 2016-07-15 | 2016-10-12 | 中南大学湘雅三医院 | Non-diagnosis-targeted HIV antibody immunity detecting method and kit |
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2016
- 2016-11-03 CN CN201610954271.XA patent/CN106546570A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297931A (en) * | 1999-11-29 | 2001-06-06 | 上海博容基因开发有限公司 | Human ABC transfer protein 39 as one new kind of polypeptide and polynucleotides encoding this polypeptide |
| CN101320042A (en) * | 2008-06-06 | 2008-12-10 | 天津市宝坻区人民医院 | Detection method and reagent kit of anti-keratin antibody |
| CN201828517U (en) * | 2010-09-25 | 2011-05-11 | 天津市宝坻区人民医院 | Kit for detecting hand-foot-and-mouth disease by adopting biotin fluorescence method |
| CN201892679U (en) * | 2010-09-25 | 2011-07-06 | 天津市宝坻区人民医院 | Influenza A H1N1 immunofluorescence detection reagent box |
| CN102532298A (en) * | 2012-03-01 | 2012-07-04 | 刘林林 | ABCC3 antigen polypeptide specially binding with autoantibody and application |
| CN204188622U (en) * | 2014-10-09 | 2015-03-04 | 天津市宝坻区人民医院 | Antikeratin antibody detection kit |
| CN105759029A (en) * | 2016-02-24 | 2016-07-13 | 天津市宝坻区人民医院 | Detection kit for measles virus and application method of detection kit |
| CN106018805A (en) * | 2016-07-15 | 2016-10-12 | 中南大学湘雅三医院 | Non-diagnosis-targeted HIV antibody immunity detecting method and kit |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110806478A (en) * | 2019-11-24 | 2020-02-18 | 天津市宝坻区人民医院 | Clostridium difficile detection kit |
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Application publication date: 20170329 |