CN108226474A - The ELISA detection kit and its detection method of GluR3B antibody - Google Patents
The ELISA detection kit and its detection method of GluR3B antibody Download PDFInfo
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- CN108226474A CN108226474A CN201711468262.0A CN201711468262A CN108226474A CN 108226474 A CN108226474 A CN 108226474A CN 201711468262 A CN201711468262 A CN 201711468262A CN 108226474 A CN108226474 A CN 108226474A
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to a kind of ELISA detection kit of GluR3B antibody, including：The ELISA ELISA Plates that are coated with GluR3B peptide fragments, the ELISA ELISA Plates for being coated with the PBS solution containing 1%BSA, ELIAS secondary antibody, standard protein, coating buffer solution, confining liquid, ELISA ELISA Plates cleaning solution, dilution, developing solution, terminate liquid.The detection kit can be used for being detected the content of GluR3B antibody in epileptic's serum, cerebrospinal fluid, detection method is easy to operate, accuracy is high, high sensitivity, and complex instrument is not needed to when detecting, it is easy to promote and apply in research institutions and medical institutions, clinical samples can be detected on a large scale, the relevant mass data of people's GluR3B antibody and information are quickly obtained, the diagnosis for Cognitive Function in Patients with Epilepsy obstacle provides important clinical reference value.
Description
Technical field
The present invention relates to a kind of ELISA detection kit and its detection method of GluR3B antibody, in immunology and biology
Technical field.
Technical background
Epilepsy is neurology department's common disease, and there are about 5,000 ten thousand epileptics now, account for about the 0.5-1% of world population.Epilepsy is suffered from
Person is often associated with the exception in terms of severe cognitive aspect, behavior, and such as attention deficit hyperactivity disorder (ADD, ADHD), emotional handicap is learned
Practise and the defect of memory, these complication may compared with epileptic attack the influence bigger to Quality of Life of Epilepsy Patients in itself.Therefore,
Find Cognitive Function in Patients with Epilepsy obstacle biomarker and intervened to improve Quality of Life of Epilepsy Patients meaning weight
Greatly.
At present, GluR3B antibody is found to be present in different types of epileptic's body of significant proportion, therefore suspects
They have brain pathogenic influence.It is further found by zoopery, in three behaviors test, (swing arm is tested, spacious field
Experiment and handgrip exercise) in, mouse generates the GluR3B antibody of high level specificity and non-specific cross-reaction (by GluR3B
Generation is immunized in peptide) abnormal behaviour and dyskinesia can be caused, and several groups of control groups (do not generate the mouse of antibody or generate high level
Other people antibody in order to control mouse, generated by the antibody mediated immunity artificially controlled), then do not show such exception.This says
Bright, spirit/cognition/abnormal behavior that GluR3B antibody is showed with epileptic is closely related.
Since GluR3B antibody can combine neuron, there is unique capability activation their antigen (respective paddy ammonia
Acid acceptor), nerve cell is killed, thus multiple cerebral injury and inducing neural and cognition/emotional handicap are caused, GluR3B antibody
As the biomarker of Cognitive Function in Patients with Epilepsy obstacle, there is great meaning for the diagnosis of Cognitive Function in Patients with Epilepsy obstacle
Justice, therefore, for having before epileptics, especially routine treatment invalid intractable epilepsy disease and big cerebral surgery operation
Necessity detection GluR3B peptide antibodies, but at present, there is no literature reported on the ELISA detection kit of GluR3B antibody, also without needle
A kind of accurate, easy, high sensitivity detection means is provided the GluR3B antibody contents in epileptic's serum.
Invention content
Present invention aims to solve the deficiencies of the prior art, and provides a kind of a kind of ELISA detection kit of GluR3B antibody,
It can be used for being detected the content of GluR3B antibody in epileptic's serum, cerebrospinal fluid.
Another object of the present invention also reside in provide using the ELISA detection kit to the content of GluR3B antibody into
The method of row detection, this method is simple to operate, and accuracy is high, high sensitivity.
To solve the above-mentioned problems of the prior art, the technical solution that the present invention takes is：
A kind of ELISA detection kit of GluR3B antibody, including：
A. the ELISA ELISA Plates of GluR3B peptide fragments are coated with, GluR3B peptide fragments biotechnology of being shone by force by Shanghai has
Limit company synthesizes, and sequence is：NEYERFVPFSDQQISNDSSSSENR, coating peptide fragment working concentration are 20ug/ml；
B. the ELISA ELISA Plates of the PBS solution containing 1%BSA are coated with；This ELISA Plate is to exclude resisting for non-specific binding
Body；
C. ELIAS secondary antibody, the Goat anti-Human IgG for HRP labels；Purchased from Jackson ImmunoResearch
D. standard protein is GluR3B monoclonal antibodies；
E. buffer solution, confining liquid, ELISA ELISA Plates cleaning solution, dilution, developing solution, terminate liquid are coated with.
The coating pH of buffer is 9.6, preparation method：By 0.16g Na2CO3With 0.29g NaHCO3Use distilled water
Dissolving, is then settled to 100mL.
The preparation method of the confining liquid：1g BSA are dissolved in PBS solution, are then settled to 100mL.
The preparation method of the ELISA ELISA Plates cleaning solution：Take 3.75g Na2HPO4·12H2O、0.43g NaH2PO4·
2H2O and 7.2g NaCl are settled to 1L after being dissolved using distilled water.
The same confining liquid of preparation method of the dilution.
The developing solution is ABTS peroxidase substrates, purchased from Kirkegaard＆Perry Laboratories.
The terminate liquid is the sulfuric acid solution of 2mol/l.
The method being detected using the ELISA detection kit to the content of GluR3B antibody, is included the following steps：
(1) GluR3B peptide fragments are diluted to 20ug/ml using coating buffer solution, 100ul are added in every hole of ELISA Plate,
Sealing plate is placed on 4 DEG C of overnight incubations, spare；Confining liquid is added in per hole in another ELISA Plate, similary sealing plate is placed on 4 DEG C and was incubated
It is night, spare；
(2) next day gets rid of the liquid in each hole, adds in ELISA ELISA Plate cleaning solution 300ul, washs 3-5 times, dries, will
Confining liquid is loaded onto with 100ul/ pore volumes on two pieces of ELISA Plates, is incubated at room temperature 2 hours, is got rid of the liquid in each hole, is added in
ELISA ELISA Plate cleaning solution 300ul are washed 3-5 times, drying；
(3) epileptic's serum or CSF sample are loaded onto the corresponding hole of two pieces of ELISA Plates with 100ul/ pore volumes
On, standard protein, into various concentration gradient, the corresponding hole of two pieces of ELISA Plates is loaded onto with 100ul/ pore volumes through diluted
On, 4 DEG C of incubated overnights of ELISA Plate, next day, drying adds in ELISA ELISA Plate cleaning solution 300ul, washs 3-5 times, dries；
(4) by ELIAS secondary antibody dilution with 1：Then 1000 dilution proportion is loaded onto two pieces with 100ul/ pore volumes
On ELISA Plate, ELISA Plate is incubated at room temperature 2 hours, gets rid of the liquid in each hole, adds in ELISA ELISA Plate cleaning solution 300ul, washing
3-5 times, drying；
(5) developing solution is added in, room temperature is protected from light incubation 10-20 minutes, adds in terminate liquid and terminates reaction, in microplate reader
405nm measures OD values.
Final OD values are calculated using the following formula：[GluR3B coated elisa plate OD values]-[confining liquid coated elisa plate OD
Value]
Advantageous effect：The ELISA detection kit of humanized's GluR3B antibody of commercial-free currently on the market, the present invention
The ELISA detection kit of GluR3B antibody can accurately detect the content of GluR3B antibody in epileptic's serum, cerebrospinal fluid,
As a result by microplate reader quantitative analysis, the subjectivity of the semi-quantitative methods such as immunohistochemistry, immunofluorescence is eliminated；High sensitivity is used
The minimum 10ng/ml of GluR3B antibody that this method detects, sensitivity are apparently higher than common Western blot and immune group
The semi-quantitative methods such as change；Agents useful for same and experiment consumptive material are commercially available commercially produced product in the LISA detection kits of the present invention,
It is easy to get, detection method is simple and convenient, and pipettor is only needed in detection and microplate reader is loaded and reading, common laboratory
It can carry out this detection with hospital, a kind of new means and method are provided for clinical examination and basic research.
The GluR3B antibody that the kit of the present invention is mainly used in the serum of epileptic, spinal fluid samples is quantitatively examined
It surveys, the GluR3B that can operate in basic research in various biological samples (such as cell culture supernatant, cell pyrolysis liquid) resists
The detection of body.Complex instrument is not needed to during detection, is easy to promote and apply in research institutions and medical institutions, can detect on a large scale
Clinical samples are quickly obtained the relevant mass data of people's GluR3B antibody and information, for examining for Cognitive Function in Patients with Epilepsy obstacle
It is disconnected that important clinical reference value is provided, have a vast market prospect, larger economic and social benefit.
Specific embodiment
With reference to specific embodiment, the invention will be further described.
Embodiment 1
A kind of ELISA detection kit of GluR3B antibody, including：
A. the ELISA ELISA Plates of GluR3B peptide fragments are coated with, GluR3B peptide fragments biotechnology of being shone by force by Shanghai has
Limit company synthesizes, and sequence is：NEYERFVPFSDQQISNDSSSSENR, coating peptide fragment working concentration are 20ug/ml；
B. the ELISA ELISA Plates of the PBS solution containing 1%BSA are coated with；This ELISA Plate is to exclude resisting for non-specific binding
Body；
C. ELIAS secondary antibody, the Goat anti-Human IgG for HRP labels；Purchased from Jackson ImmunoResearch；
D. standard protein is GluR3B monoclonal antibodies；
E. buffer solution, confining liquid, ELISA ELISA Plates cleaning solution, dilution, developing solution, terminate liquid are coated with.
The coating pH of buffer is 9.6, preparation method：By 0.16g Na2CO3With 0.29g NaHCO3Use distilled water
Dissolving, is then settled to 100mL.
The preparation method of the confining liquid：1g BSA are dissolved in PBS solution, are then settled to 100mL.
The preparation method of the ELISA ELISA Plates cleaning solution：Take 3.75g Na2HPO4·12H2O、0.43g NaH2PO4·
2H2O and 7.2g NaCl are settled to 1L after being dissolved using distilled water.
The same confining liquid of preparation method of the dilution.
The developing solution is ABTS peroxidase substrates, purchased from Kirkegaard＆Perry Laboratories.
The terminate liquid is the sulfuric acid solution of 2mol/l.
The method being detected using above-mentioned ELISA detection kit to the content of GluR3B antibody, is included the following steps：
(1) GluR3B peptide fragments are diluted to 20ug/ml using coating buffer solution, 100ul are added in every hole of ELISA Plate,
Sealing plate is placed on 4 DEG C of overnight incubations, spare；Confining liquid is added in per hole in another ELISA Plate, similary sealing plate is placed on 4 DEG C and was incubated
It is night, spare；
(2) next day gets rid of the liquid in each hole, adds in ELISA ELISA Plate cleaning solution 300ul, washs 3-5 times, dries, will
Confining liquid is loaded onto with 100ul/ pore volumes on two pieces of ELISA Plates, is incubated at room temperature 2 hours, is got rid of the liquid in each hole, is added in
ELISA ELISA Plate cleaning solution 300ul are washed 3-5 times, drying；
(3) epileptic's serum or CSF sample are loaded onto the corresponding hole of two pieces of ELISA Plates with 100ul/ pore volumes
On, standard protein, into various concentration gradient, the corresponding hole of two pieces of ELISA Plates is loaded onto with 100ul/ pore volumes through diluted
On, 4 DEG C of incubated overnights of ELISA Plate, next day, drying adds in ELISA ELISA Plate cleaning solution 300ul, washs 3-5 times, dries；
(4) by ELIAS secondary antibody dilution with 1：Then 1000 dilution proportion is loaded onto two pieces with 100ul/ pore volumes
On ELISA Plate, ELISA Plate is incubated at room temperature 2 hours, gets rid of the liquid in each hole, adds in ELISA ELISA Plate cleaning solution 300ul, washing
3-5 times, drying；
(5) developing solution is added in, room temperature is protected from light incubation 10-20 minutes, adds in terminate liquid and terminates reaction, in microplate reader
405nm measures OD values.
First, sensitivity experiments
By standard protein, i.e. GluR3B monoclonal antibodies, with confining liquid doubling dilution, diluted concentration 10ng/ml, 3ng/
Ml, 1ng/ml, 0.3ng/ml, 0.1ng/ml, 0.03ng/ml, 0.01ng/ml, each concentration standard albumen set 2 multiple holes,
Blank group uses confining liquid, is tested according to the step of above-mentioned detection method, 0.01ng/ml concentration standard protein groups OD
Value is different statistically significant with blank group OD value differences, the experimental results showed that detectable minimum concentration is 10ng/ml.
2nd, repeatability experiment
It by standard protein, i.e. GluR3B monoclonal antibodies, is diluted with confining liquid, diluted concentration 250ng/ml,
125ng/ml, 62.50ng/ml, 31.25ng/ml, 15.63ng/ml.According to the step 3 repeated experiments of above-mentioned detection method,
First time experimental result：1.967,1.147,0.660,0.418,0.309；Second of experimental result：1.971,1.159,
0.672,0.422,0.286；Third time experimental result：1.964,1.117,0.597,0.366,0.20, it is poor between three groups of OD values
It is different not statistically significant, show to stablize using the kit testing result.
Claims (7)
1. a kind of ELISA detection kit of GluR3B antibody, which is characterized in that including：
A. the ELISA ELISA Plates of GluR3B peptide fragments are coated with, coating peptide fragment working concentration is 20ug/ml；
B. the ELISA ELISA Plates of the PBS solution containing 1%BSA are coated with；
C. ELIAS secondary antibody, the Goat anti-Human IgG for HRP labels；
D. standard protein is GluR3B monoclonal antibodies；
E. buffer solution, confining liquid, ELISA ELISA Plates cleaning solution, dilution, developing solution, terminate liquid are coated with.
2. the ELISA detection kit of GluR3B antibody as described in claim 1, which is characterized in that the coating buffer solution
PH is 9.6, preparation method：By 0.16gNa2CO3With 0.29g NaHCO3It is dissolved with distilled water, is then settled to 100mL.
3. the ELISA detection kit of GluR3B antibody as described in claim 1, which is characterized in that the system of the confining liquid
Preparation Method：1gBSA is dissolved in PBS solution, is then settled to 100mL.
4. the ELISA detection kit of GluR3B antibody as described in claim 1, which is characterized in that the ELISA ELISA Plates
The preparation method of cleaning solution：Take 3.75g Na2HPO4·12H2O、0.43g NaH2PO4·2H2O and 7.2g NaCl use double steamings
1L is settled to after water dissolution.
5. the ELISA detection kit of GluR3B antibody as described in claim 1, which is characterized in that the developing solution is
ABTS peroxidase substrates.
6. such as the ELISA detection kit of GluR3B antibody described in any one of claim 1 to 5, which is characterized in that the end
Only liquid is the sulfuric acid solution of 2mol/l.
7. the side being detected using any one of claim 1 to 6 ELISA detection kit to the content of GluR3B antibody
Method, which is characterized in that include the following steps：
(1) GluR3B peptide fragments are diluted to 20ug/ml using coating buffer solution, 100ul, sealing plate is added in every hole of ELISA Plate
4 DEG C of overnight incubations are placed on, it is spare；Confining liquid is added in per hole in another ELISA Plate, similary sealing plate is placed on 4 DEG C of overnight incubations,
It is spare；
(2) next day gets rid of the liquid in each hole, adds in ELISA ELISA Plate cleaning solution 300ul, washs 3-5 times, and drying will be closed
Liquid is loaded onto with 100ul/ pore volumes on two pieces of ELISA Plates, is incubated at room temperature 2 hours, gets rid of the liquid in each hole, adds in ELISA enzymes
Target cleaning solution 300ul is washed 3-5 times, drying；
(3) epileptic's serum or CSF sample are loaded onto with 100ul/ pore volumes on the corresponding hole of two pieces of ELISA Plates, marked
Quasi- albumen, into various concentration gradient, is loaded onto with 100ul/ pore volumes on the corresponding hole of two pieces of ELISA Plates, enzyme through diluted
4 DEG C of incubated overnights of target, next day, drying add in ELISA ELISA Plate cleaning solution 300ul, wash 3-5 times, drying；
(4) by ELIAS secondary antibody dilution with 1：Then 1000 dilution proportion is loaded onto two pieces of enzyme marks with 100ul/ pore volumes
On plate, ELISA Plate is incubated at room temperature 2 hours, gets rid of the liquid in each hole, adds in ELISA ELISA Plate cleaning solution 300ul, washs 3-5
It is secondary, drying；
(5) developing solution is added in, room temperature is protected from light incubation 10-20 minutes, adds in terminate liquid and terminates reaction, 405nm is surveyed in microplate reader
Determine OD values.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1945335A (en) * | 2005-10-28 | 2007-04-11 | 中国科学院上海生命科学研究院 | Reagent kit for detecting hepatitis B virus e antigen and use |
| US20140018376A1 (en) * | 2009-10-20 | 2014-01-16 | Hans Allgeier | Use of 1H-Quinazoline-2,4-Diones |
-
2017
- 2017-12-29 CN CN201711468262.0A patent/CN108226474A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1945335A (en) * | 2005-10-28 | 2007-04-11 | 中国科学院上海生命科学研究院 | Reagent kit for detecting hepatitis B virus e antigen and use |
| US20140018376A1 (en) * | 2009-10-20 | 2014-01-16 | Hans Allgeier | Use of 1H-Quinazoline-2,4-Diones |
Non-Patent Citations (2)
| Title |
|---|
| HADASSA GOLDBERG-STERN ET AL.: "Glutamate receptor antibodies directed against AMPA receptors subunit 3 peptide B (GluR3B) associate with some cognitive/psychiatric/behavioral abnormalities in epilepsy patients", 《PSYCHONEUROENDOCRINOLOGY》 * |
| 李昕昱 等: "癫痫患者脑脊液中多巴胺的改变及意义", 《徐州医科大学学报》 * |
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