CN108535494A - A kind of IL-6ELISA detection kits and its application method - Google Patents
A kind of IL-6ELISA detection kits and its application method Download PDFInfo
- Publication number
- CN108535494A CN108535494A CN201810712991.4A CN201810712991A CN108535494A CN 108535494 A CN108535494 A CN 108535494A CN 201810712991 A CN201810712991 A CN 201810712991A CN 108535494 A CN108535494 A CN 108535494A
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- China
- Prior art keywords
- kit according
- antibody
- hrp
- elisa plate
- enzyme dilution
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6869—Interleukin
Abstract
The present invention relates to a kind of IL 6ELISA detection kits.The present invention is the principle using enzyme-linked adsorption experiment double antibody sandwich method, utilize coating 6 monoclonal antibody ELISA Plates of IL, sample and label 6 monoclonal antibody one-step method of the IL reaction of HRP enzymes is added, it is developed the color with TMB, come quantify detection IL 6 content, this experiment detection method compared with other methods operation it is easier, flux is big, it is cheap, it is suitble to extensive screening.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of IL-6ELISA detection kits and its use.
Background technology
IL-6 is mainly generated by various kinds of cell such as macrophage, T cell, B cells.The growth of its adjustable various kinds of cell with
Differentiation has and adjusts immune response, acute phase response and hematopoiesis function, and plays important work in the reaction of the anti-infectious immunity of body
With.IL-6 is substantially change in a variety of diseases.IL-6 expression imbalances can cause many diseases, clinical manifestation predominantly to be fallen ill
When IL-6 levels increase.It the level that IL-6 rises and the development and change of the active stage, tumour of disease, rejection degree and controls
Therapeutic effect is all closely related, therefore, the change of illness state of patient can be reflected to the detection of IL-6 levels in patient's body fluid.
IL-6 has been acknowledged as a kind of important proinflammatory cytokine, participates in body inflammatory reaction and anti-infectious function.It grinds
Study carefully and shows that IL-6 cooperates with the pathologic process for participating in wound and inflammation disease, research with cytokine profiles under infection conditions
Personnel and clinician think the diagnosis index that in infectious diseases IL-6 is more more sensitive than c reactive protein.
Current is mainly clinically chemiluminescence immunoassay with IL-6 detection methods, and method uses double-antibody sandwich
Method, the difference is that marker is different：Chemoluminescence method is alkaline phosphatase.Chemoluminescence method accurately, can be detected quickly
Go out as a result, still this method instrument cost is relatively high, needs specific chemiluminescence instrument, and to experiment operator
It is required that relatively high, only some relatively large laboratories can just have corresponding condition.
Therefore, clinical to need to research and develop a kind of sensibility height, high specificity, simple and convenient, inexpensive suitable clinic
The IL-6 detection methods of popularization.
Invention content
The purpose of the present invention is to provide a kind of IL-6ELISA detection kits and its application methods, to solve
Testing cost height, technical problem complicated for operation in the prior art, the operation of this patent is easier, and flux is big, cheap, fits
Close extensive screening.
To solve the above-mentioned problems, the present invention provides a kind of IL-6ELISA detection kits, which is characterized in that includes
Following components：Coating processing after ELISA Plate, HRP label IL-6 antibody, matched curve standard items, standard dilutions and A,
B developing solutions.
The invention further relates to a kind of preferred kit protocols, which is characterized in that after the coating processing in ELISA Plate
Coated antibody is：IL-6 monoclonal antibodies.
The invention further relates to a kind of preferred kit protocols, which is characterized in that after the coating processing in ELISA Plate
Closing formula of liquid be：+ 0.2% polysorbas20 of 0.02MPBS+1%BSA+0.1%l caseins sodium salt.
The invention further relates to a kind of preferred kit protocols, which is characterized in that the IL-6 antibody of the described HRP labels is
Monoclonal antibody.
The invention further relates to a kind of preferred kit protocols, which is characterized in that the standard dilutions are that enzyme is dilute
Release liquid.
The invention further relates to a kind of preferred kit protocols, which is characterized in that the enzyme dilution formula is：
+ 0.2% Triton X-100+0.1%proclin300 of 0.02MPBS+10% calf serums.
The invention further relates to a kind of preferred kit protocols, which is characterized in that the use concentration of the enzyme dilution
For：1：1000,1：2000,1：4000,1：8000.
The invention further relates to a kind of preferred kit protocols, which is characterized in that the use concentration of the enzyme dilution
For：1：2000.
The invention further relates to a kind of preferred kit protocols, which is characterized in that A, B developing solution develops the color for TMB
Liquid.
Step 1：Take the IL-6 antibody of 50 microlitres of samples and 50 microlitres of HRP enzymes label to be added coating treated ELISA Plate,
And standard items are diluted to 0pg/ml, 20pg/ml, 40pg/ml, 80pg/ml, 160pg/ml, 320pg/ml, 640pg/ml.
Step 2：4 times, which are washed, with conventional PBST washing lotions pats dry addition TMB developing solutions；
Step 3：Terminate liquid is added after being protected from light colour developing 15 minutes to terminate；
Step 4：It is read with microplate reader.
The invention further relates to a kind of preferred schemes using kit method, which is characterized in that the sample to be tested
Selected from blood plasma and serum.
The invention further relates to a kind of preferred schemes using kit method, which is characterized in that the HRP enzymes label
IL-6 antibody concentrations be 1：2000.
The invention further relates to a kind of preferred schemes using kit method, which is characterized in that the terminate liquid is
Sulfuric acid.
Description of the drawings
Attached drawing 1 is standard fit curve
Specific implementation mode
The present invention is further described with reference to embodiments, but these embodiments not limit the scope of the present invention.
Embodiment 1：ELISA Plate is coated with and closing
20ugIL-6 monoclonal antibodies (the magnificent biology in Wuhan) are taken to be added to the carbonate buffer of 10ml0.05M pH values 9.6
It is uniformly mixed, is even added in 96 hole elisa Plates in liquid, per hole 100ul, 37 degree are reacted 1 hour, and 4 degree of coatings 16 of rear placement are small
When, it pats dry, washing 3 times with conventional PBST washing lotions pats dry, and confining liquid is added, and reaction pats dry for 1 hour, 37 degree of air dry oven dryings 1
Hour, it is spare that 4 degree of refrigerators are placed in sealing.
Close formula of liquid：+ 0.2% polysorbas20 of 0.02MPBS+1%BSA+0.1%l caseins sodium salt.
Embodiment 2：HRP enzymes mark IL-6 monoclonal antibodies：
HRP enzymes are dissolved into ultra-pure water, its final concentration of 10mg/ml is made, 1ml is taken to take 500ml10mg/ml again, 4 degree anti-
It answers 1 hour, adds 500ul1% ethylene glycol and be uniformly mixed, 4 degree are reacted 1 hour, take 250ul to be added in final product
5mgIL-6 monoclonal antibodies (the magnificent biology in Wuhan) are uniformly mixed, and are dialysed 16 hours in 0.05MPH9.6 carbonate buffer solutions,
Midway change a not good liquor, add 30ul5mg/ml sodium borohydrides terminate reaction, 4 degree 2 hours, with G25 desalting columns centrifuge desalination obtain
It is HRP-IL-6 enzyme markers to final product, 50% glycerine is added and places -20 degree preservations.
Developing solution is prepared：
A liquid：Sodium acetate 12.6g+ citric acid 1.6g+ urea peroxides 0.3g is settled to 500ml；
B liquid：EDTA-2Na0.2g+ citric acids 0.95g+50ml glycerine+tetramethyl biphenyl diamines 0.2g (is dissolved in 1mlDMSO
In) it is settled to 500ml.
Standard items are prepared：After IL-6 antigens chemiluminescence assignment, it is diluted to 640pg/ml (1ml) with enzyme dilution and does
Initial concentration.
Standard dilutions：+ 0.2% Triton X-100+0.1%proclin300 of 0.02MPBS+10% calf serums.
Embodiment 3：The determination of enzyme dilution optimal use concentration
Enzyme dilution formula：+ 0.2% Triton X-100+0.1%proclin300 of 0.02MPBS+10% calf serums.
The determination of best HRP-IL-6：
Product in embodiment 2 is diluted to 1 respectively with enzyme dilution is dilute：1000,1：2000,1：4000,1：8000；
IL-6 antigens are diluted to 20pg/ml, 1000pg/ml with enzyme dilution, make upper negative control respectively of dilution
And parallel hole is done, take 50ul that 1 ready-made production board of embodiment is added, then be respectively separately added into HRP-IL-61：1000,1：2000,
1：4000,1：8000, each 50ul, 37 degree are reacted 30 minutes, and washing 4 times with conventional PBST washing lotions pats dry addition TMB developing solution (routines
Kit) be protected from light colour developing 15 minutes after be added terminate liquid (2M sulfuric acid) terminate.It is read respectively with 450nm wavelength microplate reader.
As a result as follows：
As a result illustrate, 1：The OD values of 2000 enzymes are bigger, and background is relatively low so enzyme optimal use a concentration of 1：2000.
Embodiment 4：The application method of kit
IL-6 antigens (the magnificent biology in Wuhan) Roche E610 chemiluminescence instrument assignment is taken, is diluted respectively with enzyme dilution
It is standard items initial value at 640pg/ml.And standard items are diluted to successively：0pg/ml, 20pg/ml, 40pg/ml, 80pg/ml,
160pg/ml, 320pg/ml, 640pg/ml.The sample 10 after Roche E610 chemiluminescence detection assignment is taken, sampling is originally each
50ul is added, adds 1:2000 HRP-IL-6 enzymes 50ul, each sample do multiple holes, and 37 degree are reacted 30 minutes, with routine
PBST washing lotions wash 4 times and pat dry addition TMB developing solutions, and terminate liquid (2M sulfuric acid), which is added, after being protected from light colour developing 15 minutes terminates, and uses 450nm
Wavelength microplate reader is read respectively.
Curve such as attached drawing 1 is obtained using above-mentioned experimental data
Four parameter Logistic Fitting curve equations：Y=(A-D)/[1+ (x/C) ^B]+D
A=2.49472
B=-1.59145
C=117.22045
D=0.09187
R^2=0.99976
==================================
The experimental results showed that：Within 10 samples and chemiluminescence assignment deviation all 10%, illustrate its accuracy, with shine
As a result highly consistent, it can be tested with clinical detection.
Claims (10)
1. a kind of IL-6ELISA detection kits, which is characterized in that include following components：ELISA Plate, HRP marks after coating processing
The IL-6 antibody of note, the standard items of matched curve, standard items enzyme dilution and A, B developing solution.
2. kit according to claim 1, which is characterized in that coated antibody in ELISA Plate after the coating processing
For：IL-6 monoclonal antibodies.
3. kit according to claim 1, which is characterized in that the confining liquid after the coating processing in ELISA Plate is matched
Fang Wei：+ 0.2% polysorbas20 of 0.02MPBS+1%BSA+0.1% caseins sodium salt.
4. kit according to claim 1, which is characterized in that the IL-6 antibody of the HRP labels is anti-for monoclonal
Body.
5. kit according to claim 1, which is characterized in that the standard dilutions are enzyme dilution.
6. kit according to claim 5, which is characterized in that the enzyme dilution formula is：0.02MPBS+10%
+ 0.2% Triton X-100+0.1%proclin300 of calf serum.
7. kit according to claim 5, which is characterized in that the use of the enzyme dilution is a concentration of：1:1000,
1:2000,1:4000,1:8000, preferably 1:2000.
8. kit according to claim 1, which is characterized in that A, B developing solution is TMB developing solutions.
9. a kind of application method of IL-6ELISA detection kits, it is characterised in that：
Step 1：Take the IL-6 antibody of 50 microlitres of samples to be tested and 50 microlitres of HRP enzymes label to be added coating treated ELISA Plate,
And standard items are diluted to 0pg/ml, 20pg/ml, 40pg/ml, 80pg/ml, 160pg/ml, 320pg/ml, 640pg/ml, often
Hole adds 100 microlitres.
Step 2：4 times, which are washed, with conventional PBST washing lotions pats dry addition TMB developing solutions；
Step 3：Terminate liquid is added after being protected from light colour developing 15 minutes to terminate；
Step 4：It is read with microplate reader.
10. according to the method described in claim 10, it is characterized in that, the sample to be tested is selected from blood plasma and serum；It is described
HRP enzymes label IL-6 antibody concentrations be 1:2000；The terminate liquid is sulfuric acid.
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| CN201810712991.4A CN108535494A (en) | 2018-06-29 | 2018-06-29 | A kind of IL-6ELISA detection kits and its application method |
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| CN201810712991.4A CN108535494A (en) | 2018-06-29 | 2018-06-29 | A kind of IL-6ELISA detection kits and its application method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112269025A (en) * | 2020-11-13 | 2021-01-26 | 三诺生物传感股份有限公司 | Interleukin-6 chemiluminescence assay kit and preparation method thereof |
| CN113376380A (en) * | 2021-05-31 | 2021-09-10 | 华南农业大学 | ELISA kit for detecting dog IL-6 and application thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005108986A1 (en) * | 2004-05-10 | 2005-11-17 | Korea Research Institute Of Bioscience And Biotechnology | A diagnostic method of asthma using baff as a diagnostic marker |
| EP1837658A1 (en) * | 2006-03-22 | 2007-09-26 | Paul-Ehrlich-Institut Bundesamt für Sera und Impfstoffe | Cytokine-based pyrogen test |
| CN101393204A (en) * | 2008-10-16 | 2009-03-25 | 中山大学 | Applications of blood serum IL-6 for preparing diagnose kit for primary hepatocellular carcinoma |
| CN102393456A (en) * | 2011-08-24 | 2012-03-28 | 北京正旦国际科技有限责任公司 | Kit for detecting Hepassocin (HPS) |
| CN103969438A (en) * | 2014-04-29 | 2014-08-06 | 北京普恩光德生物科技开发有限公司 | Detection kit for interleukin 6 |
| CN104198728A (en) * | 2014-08-22 | 2014-12-10 | 广西南宁隆吉维特生物科技有限公司 | Human serum CXCL16 enzyme-linked immunosorbent assay kit as well as preparation and use methods thereof |
| CN106093432A (en) * | 2016-06-13 | 2016-11-09 | 厦门大学 | Based on joint-detection FGF1, the kit for breast cancer of FGF10 and IL6 |
| CN106645734A (en) * | 2015-11-02 | 2017-05-10 | 博格·孙杨 | Septin9 detection kit based on enzyme linked immunosorbent assay |
| CN106706912A (en) * | 2015-07-21 | 2017-05-24 | 中国科学院上海生命科学研究院 | Marker for diagnosis of inflammation-associated HCC and application thereof |
| US20180095077A1 (en) * | 2016-11-27 | 2018-04-05 | Alireza Palangi | Quality control system and kit for automated elisa devices |
-
2018
- 2018-06-29 CN CN201810712991.4A patent/CN108535494A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005108986A1 (en) * | 2004-05-10 | 2005-11-17 | Korea Research Institute Of Bioscience And Biotechnology | A diagnostic method of asthma using baff as a diagnostic marker |
| EP1837658A1 (en) * | 2006-03-22 | 2007-09-26 | Paul-Ehrlich-Institut Bundesamt für Sera und Impfstoffe | Cytokine-based pyrogen test |
| CN101393204A (en) * | 2008-10-16 | 2009-03-25 | 中山大学 | Applications of blood serum IL-6 for preparing diagnose kit for primary hepatocellular carcinoma |
| CN102393456A (en) * | 2011-08-24 | 2012-03-28 | 北京正旦国际科技有限责任公司 | Kit for detecting Hepassocin (HPS) |
| CN103969438A (en) * | 2014-04-29 | 2014-08-06 | 北京普恩光德生物科技开发有限公司 | Detection kit for interleukin 6 |
| CN104198728A (en) * | 2014-08-22 | 2014-12-10 | 广西南宁隆吉维特生物科技有限公司 | Human serum CXCL16 enzyme-linked immunosorbent assay kit as well as preparation and use methods thereof |
| CN106706912A (en) * | 2015-07-21 | 2017-05-24 | 中国科学院上海生命科学研究院 | Marker for diagnosis of inflammation-associated HCC and application thereof |
| CN106645734A (en) * | 2015-11-02 | 2017-05-10 | 博格·孙杨 | Septin9 detection kit based on enzyme linked immunosorbent assay |
| CN106093432A (en) * | 2016-06-13 | 2016-11-09 | 厦门大学 | Based on joint-detection FGF1, the kit for breast cancer of FGF10 and IL6 |
| US20180095077A1 (en) * | 2016-11-27 | 2018-04-05 | Alireza Palangi | Quality control system and kit for automated elisa devices |
Non-Patent Citations (2)
| Title |
|---|
| 印莉萍 等: "酶联免疫吸附测定", 《分子细胞生物学实验技术》 * |
| 吕世静: "酶标记物的制备", 《临床免疫学检验》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112269025A (en) * | 2020-11-13 | 2021-01-26 | 三诺生物传感股份有限公司 | Interleukin-6 chemiluminescence assay kit and preparation method thereof |
| CN113376380A (en) * | 2021-05-31 | 2021-09-10 | 华南农业大学 | ELISA kit for detecting dog IL-6 and application thereof |
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Application publication date: 20180914 |