CN102608332A - Enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10 - Google Patents
Enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10 Download PDFInfo
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- CN102608332A CN102608332A CN2012100767688A CN201210076768A CN102608332A CN 102608332 A CN102608332 A CN 102608332A CN 2012100767688 A CN2012100767688 A CN 2012100767688A CN 201210076768 A CN201210076768 A CN 201210076768A CN 102608332 A CN102608332 A CN 102608332A
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Abstract
The invention relates to a competitive inhibition enzyme-linked immunoassay (ELISA) method of grass carp interleukin 10 (IL-10). The method comprises the following steps of: immunizing a New Zealand white rabbit by utilizing grass carp interleukin 10 proteins which is expressed in a recombined way as an immunogen to obtain a polyclonal antibody; marking the antibody by utilizing horseradish peroxidase; and finally, establishing the competitive inhibition ELISA method by taking the recombined grass carp interleukin IL-10 protein as a covered antigen, wherein the competitive inhibition ELISA method can be used for the protection detection of the grass carp interleukin 10. The detection range of the ELISA method is 0.1-100 ng/mL. By utilizing the polyclonal antibody and the competitive inhibition ELISA method, the cost is low, and the stability and the repeatability are good. The conventional method is that the competitive inhibition ELISA method is firstly established to detect protection expression of the grass carp interleukin 10.
Description
Technical field
The invention belongs to enzyme linked immunosorbent detection (ELISA) biological technical field, be specifically related to a kind of grass carp interleukin 10 competition inhibitory enzyme linked immune detection method.
Background technology
(interleukin-10 IL-10) is a kind of important cytokine to interleukin 10.Discovery mouse Th2 cells such as Fiorentino in 1989 can be secreted a kind of material that suppresses cell factors such as Thl emiocytosis interferon; Therefore with this material called after cytokine secretion inhibiting factor (cytokine synthesis inhibiting factor; CSIF), be called interleukin 10 after.Discovering subsequently, this cell factor plays an important role to inflammation, malignant tumour, autoimmune disease.The increment differentiation of its ability suppressor T cell, macrophage also can promote the stable differentiation of B cell simultaneously, and its immunological diversity plays an important role it in immune system.Present research shows that also interleukin 10 has immunoloregulation function and possible therapeutic action for struvite animal.
Grass carp (Ctenopharyngodon idellus) belongs to Cypriniformes Cyprinidae graining subfamily grass carp and belongs to, and has been commonly called as: grass carp, oily grass carp, careless grass carp, ctenopharyngodon idellus, grass carp, grass roots (northeast), charlatan, black black carp etc.English name: Grass carp.Because of its growth is rapid, feed resource is wide, is one of four large Chinese carps of CHINESE FRESHWATER breed, has important economic value.But grass carp premunition and survival rate are lower; Be prone to suffer from hemorrhage, fin rot, red skin disease and enteritis disease etc.; High-density breeding has increased the chance of cross infections between the aquatic livestock in addition; Disease is increasingly sharpened, and it is very necessary therefore to study immune all kinds of immune responses of grass carp and mechanism thereof.Interleukin 10 has extensive BA in the fish immunity reaction; Requisite effect is being brought into play in immune regulation and control; Therefore the secretion situation that detects IL-10 in the grass carp not only helps understanding the grass carp immune regulation mechanism, and is that the disease prevention and cure of carrying out these fish provide useful information.But; The secretion detection method of fish IL-10 is not set up at present; Main because the IL-10 molecular structure of the IL-10 amphibian of bony fish IL-10 and mammal, amphibian differs greatly, can not use other ethnic IL-10 antigens and antibody thereof to replace analyzing and testing bony fish IL-10.The shortage of fish IL-10 antibody makes the secretion detection of IL-10 and the meticulous evaluation of function is absorbed in bottleneck simultaneously.This research prepares polyclonal antibody through recombinant protein grass carp IL-10, and successfully sets up the ELISA detection system of IL-10, for the secretion that detects IL-10 and illustrate its function status method that provides the foundation.
Summary of the invention
The objective of the invention is to set up grass carp interleukin 10 ELISA detection method, can be used for the detection of grass carp IL-10 protein expression level, owing to adopt polyclonal antibody, expense is lower and have good stable property and repeatability, detects and is limited to 0.1-100ng/mL.
To achieve these goals; The present invention utilizes recombinant expressed grass carp interleukin 10 to obtain polyclonal antibody as the immunogen immune healthy rabbit; Use this polyclonal antibody of horseradish peroxidase (HRP) mark again, set up competition inhibitory enzyme linked immune detection method with the polyclonal antibody of reorganization grass carp interleukin 10 albumen and HRP mark at last.
Technique scheme may further comprise the steps:
1) preparation of antigen protein
With grass carp IL-10cDNA sequence clone in prokaryotic expression carrier pET-30a (+); Make up recombinant prokaryotic expression vector; And this carrier is converted into carries out prokaryotic expression (Zhou Hong, Wei He, Wang Xinyan in the e. coli bl21 (DE3); A kind of grass carp interleukin 10 gene and albumen and recombinant expression method thereof, one Chinese patent application number: 201110096027.1).The gained recombinant protein is antigen protein.
2) grass carp interleukin 10 Polyclonal Antibody Preparation
With reorganization grass carp interleukin 10 albumen is antigen, and immune new zealand white rabbit also utilizes polyclonal antibody preparation and purification technique to obtain grass carp interleukin 10 polyclonal antibody, this antibody can with special the combining of grass carp interleukin 10.
3) horseradish peroxidase-labeled of grass carp interleukin 10 polyclonal antibody
Adopt horseradish peroxidase kit (available from Thermo Scientific company) the mark grass carp IL-10 polyclonal antibody of maleimide activation.
4) ELISA method of operating
The righttest extension rate of the grass carp interleukin 10 polyclonal antibody of horseradish peroxidase-labeled is 1: 2000 when (1) confirming that through the chessboard titrimetry ELISA detects, and the concentration of envelope antigen is 2 μ g/mL, every hole 100 μ L.
(2) encapsulate: carbonate buffer solution dilution grass carp interleukin-11 0 recombinant protein to the 2 μ g/mL with 0.05M adds in the ELISA Plate every hole 100 μ l as coating antigen with this; Dry liquid in the hole after 4 ℃ of incubated overnight; And with PBST washing three times, each 300 μ l/ holes, 3 minutes are each.
(3) sealing: with confining liquid (5% skimmed milk power, the 0.3%BSA solution of PBS dilution) room temperature sealing two hours, 300 μ l/ holes.
(4) hatching of enzyme labelled antibody and determined antigen: be diluted to corresponding concentration (0.2ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 30ng/mL, 100ng/mL, 200ng/mL) as standard items with antigen/antibody dilution (PBST) the grass carp recombinant il-10 albumen of will recombinating, (PBST does 10 to get each standard items 50 μ l and 50 μ lHRP-IL10Ab
3Doubly dilution) incubated at room 2 hours behind the mixing.Detection to testing sample: mix same incubated at room 2 hours with the HRP-IL10Ab of 50 μ l dilution in 1: 1000 after getting testing sample 50 μ l.Dry confining liquid after the ELISA Plate sealing is accomplished, and, add above-mentioned antigen-antibody mixed liquor, incubated at room 2 hours with 300 μ l PBST washing three times.
(5) colour developing: dry liquid in the hole, with 300 μ l PBST washing five times, each 3 minutes, the washing back is certain to be noted drying liquid in the hole, adds 100 μ l tmb substrates then, and 37 ℃ of reactions add 50 μ l 2M H after 20 minutes
2SO
4Cessation reaction.
(6) reading, result treatment: adopt ELIASA reading of data under absorbance 450nm, according to gained data computation sample inhibiting rate.Sample inhibiting rate %=negative sample light absorption value-standard items/testing sample light absorption value)/negative sample light absorption value * 100%; Be ordinate with the standard items inhibiting rate at last; The logarithm of standard items concentration is a horizontal ordinate production standard curve, with typical curve testing sample is carried out quantitative test.
Beneficial effect of the present invention: with reorganization grass carp IL-10 albumen as coating antigen; Set up the ELISA method that grass carp IL-10 detects; For the protein expression level that detects interleukin 10 in the grass carp provides detection means rapidly and efficiently, this method is reliable and stable, and cost is lower.
Description of drawings
Fig. 1 is western blotting checking grass carp interleukin 10 polyclonal antibody specificity glue figure; Swimming lane 1 is reorganization grass carp interleukin 10 among the figure; Swimming lane 2 is a grass carp head-kidney leucocyte albumen; An anti-Incubating Solution is a grass carp interleukin 10 polyclonal antibody dilution among the A figure, and an anti-Incubating Solution is control group (without the new zealand white rabbit of the reorganization grass carp interleukin 10 protein immunization) rabbit anteserum of same ratio dilution among the B figure.
Fig. 2 is the typical curve that grass carp interleukin 10 ELISA detects.
Embodiment
Below, in conjunction with accompanying drawing and specific embodiment the present invention is done detailed explanation.If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.
One: reagent:
The horseradish peroxidase kit of maleimide activation is available from Thermo Scientific company
Tetramethyl benzidine (TMB) is available from sky root biotech company
Other reagent are AR
Two: step:
1, immunogenic preparation: with grass carp IL-10cDNA sequence clone in prokaryotic expression carrier pET-30a (+); Make up recombinant prokaryotic expression vector; And this carrier is converted in the e. coli bl21 (DE3); Through 1mM IPTG abduction delivering recombinant protein IL-10, this albumen is mainly expressed with soluble form, adopts Ni
2+Affinity chromatography and the gel permeation chromatography purifying grass carp IL-10 albumen that obtains recombinating.(Zhou Hong, Wei He, Wang Xinyan, a kind of grass carp interleukin 10 gene and albumen and recombinant expression method thereof, one Chinese patent application number: 201110096027.1).
2, grass carp interleukin 10 Polyclonal Antibody Preparation
Choose the healthy new zealand white rabbit of 42 monthly ages, body weight 1.5-2kg; Male and female half and half; Per two is an experimental group; Immunogene is used physiological saline solution, is made into behind the solution of 2mg/mL and mixes with the complete Freund's adjuvant of equivalent with paddling process its emulsification, emulsification until an emulsion is splashed into do not scatter in the water float on the water surface till.Initial immunity reagent that emulsification is good is multi-point injection in the rabbit back, every injection 1mL.Carry out booster immunization behind the initial immunity 4 times, booster immunization is used incomplete Freund emulsification, and booster immunization is used intramuscular injection, and whenever at a distance from 10 days booster immunizations once, dosage is identical with initial immunity.From ear edge vein exploitating blood, adopt direct ELISA to measure antibody titer behind the last booster immunization, confirm that antibody titer can use back ear vein and heart bloodletting simultaneously acquisition antiserum, be collected in the 50mL centrifuge tube.This antiserum is carried out purifying through high speed centrifugation and after filtering, antibody is kept at contains 50% glycerine, 0.05%NaN at last
3PBS solution in ,-20 ℃ of freezing preservations.
3, the specificity of grass carp interleukin 10 polyclonal antibody checking
Collect grass carp head-kidney leucocyte total protein as testing sample; The positive contrast of reorganization grass carp interleukin 10 albumen; Detect the grass carp interleukin 10 polyclonal antibody specificity that is obtained with western blotting method, the prepared polyclonal antibody of experiment proof can specific recognition grass carp interleukin 10 albumen (like accompanying drawing 1).
4, the horseradish peroxidase-labeled of grass carp IL-10 polyclonal antibody
Adopt the horseradish peroxidase kit mark grass carp IL-10 polyclonal antibody of the maleimide activation of Thermo Scientific company.
5, ELISA course of reaction
The righttest extension rate of the grass carp interleukin 10 polyclonal antibody of horseradish peroxidase-labeled is 1: 2000 when 1) confirming that through the chessboard titrimetry ELISA detects, and the concentration of envelope antigen is 2 μ g/mL, every hole 100 μ L.
2) the grass carp interleukin 10 albumen of will recombinating is diluted to 2 μ g/mL with encapsulating damping fluid, every empty 100 μ l, 4 ℃ of incubated overnight of adding in ELISA Plate.Take out the ELISA Plate liquid in the hole that inclines next day, dry the back with PBST washing four times, each washing adds PBST 300 μ l, washs 3 minutes.
3) sealing: fully after the washing, with sealing damping fluid sealase target, 300 μ l/ holes, incubated at room 2 hours.
4) combining of enzyme labelled antibody and sample to be tested/standard items: with (the dilution in 1: 1000 of IL-10 enzyme labelled antibody; Dilution is PBST) 50 μ l and 50 μ l testing samples or 50 μ l standard items (standard items are the reorganization grass carp IL-10 albumen by the certain concentration dilution) mix, and incubated at room is 2 hours behind the abundant mixing.
5) sealing is accomplished hypsokinesis and is removed plate inner sealing liquid, and with PBST washing 3 times, each every hole adds PBST 300 μ l, washs 3 minutes.
The antigen antibody complex of hatching 6) washing back adding 3), every hole 100 μ l, incubated at room 2 hours.
7) liquid in the plate that inclines, with PBST washing 5 times, washed three minutes with 300 μ l PBST in each every hole.
8) after the abundant washing, thoroughly dry liquid in the hole, add 100 μ l TMB, 37 ℃ were reacted 20 minutes.
9) reading and result judge: the every empty 2M of adding H after accomplishing with the TMB reaction
2SO
450 μ l cessation reactions; Read light absorption value at the 450nm place at last; According to gained data computation sample inhibiting rate: (negative sample light absorption value-standard items/testing sample light absorption value)/negative sample light absorption value * 100%; According to standard items inhibiting rate and standard items concentration production standard curve (seeing accompanying drawing 2), testing sample is carried out quantitative test at last with typical curve.
Reliability for checking effect of the present invention; Carry out following evaluation: the reorganization grass carp interleukin 10 standard serial solution (0.2ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 30ng/mL, 100ng/mL, 200ng/mL) that will prepare detects by above-mentioned ELISA testing process, calculates the inhibiting rate of each concentration.With the inhibiting rate is ordinate, and the logarithm of reorganization grass carp interleukin 10 solution concentration is a horizontal ordinate, draws out typical curve.Competitive ELISA result and correlation parameter are seen table 1, and standard suppresses curve and sees accompanying drawing 2.From accompanying drawing 2 visible in the 0.1-100ng/ml scope this kit have favorable linearity.
Table 1 competition suppresses ELISA result's correlation parameter
(2) reappearance as a result
Precision test to grass carp interleukin 10 check ELISA method repeats 6 times between batch, and the light absorption value coefficient of variation (CV%) of gained variable concentrations standard solution is as shown in table 2.
The light absorption value coefficient of variation (n=6) of table 2 variable concentrations standard solution
(3) accuracy as a result
Test the accuracy that grass carp interleukin 10 ELISA detects through calculating recovery of standard addition.Get the reorganization grass carp interleukin 10 that blank cell culture medium adds different amounts, carry out ELISA and detect, calculate recovery of standard addition.The result sees table 3.
Table 3 sample recovery of standard addition
(4) specificity as a result
For verifying the specificity of this kit, carry out ELISA specificity cross reaction test to following albumen, the result is following:
The cross reaction of table 4 reorganization grass carp interleukin 10 and other albumen
Those of ordinary skill in the art will appreciate that embodiment described here is in order to help reader understanding's application of the present invention, should to be understood that protection scope of the present invention is not limited to such special statement and embodiment.Those of ordinary skill in the art can make various other various concrete distortion and combinations that do not break away from essence of the present invention according to these teachings disclosed by the invention, and these distortion and combination are still in protection scope of the present invention.
Claims (2)
1. grass carp interleukin 10 enzyme-linked immune detection method is characterized in that this method of inspection is the competition inhibitory enzyme linked immune detection method that utilizes polyclonal antibody and the reorganization grass carp interleukin 10 albumen of the anti-grass carp interleukin 10 of horseradish peroxidase (HRP) mark to set up.
2. detection method according to claim 1 is characterized in that this method may further comprise the steps:
1) preparation of antigen protein
Utilize prokaryotic expression system preparation reorganization grass carp interleukin 10 albumen as antigen protein.
2) grass carp interleukin 10 Polyclonal Antibody Preparation
With reorganization grass carp interleukin 10 albumen is antigen, and immune new zealand white rabbit also utilizes polyclonal antibody preparation and purification technique to obtain grass carp interleukin 10 polyclonal antibody, this antibody can with special the combining of grass carp interleukin 10.
3) horseradish peroxidase-labeled of grass carp interleukin 10 polyclonal antibody
Adopt the horseradish peroxidase-labeled grass carp IL-10 polyclonal antibody of maleimide activation.
4) ELISA method of operating
The righttest extension rate of the grass carp interleukin 10 polyclonal antibody of horseradish peroxidase-labeled is 1: 2000 when (1) confirming that through the chessboard titrimetry ELISA detects, and envelope antigen concentration is 2 μ g/mL, every hole 100 μ L.
(2) encapsulate: with carbonate buffer solution dilution grass carp interleukin-11 0 recombinant protein to the 2 μ g/mL of 0.05M; Add in the ELISA Plate as coating antigen with this; Every hole 100 μ l dry liquid in the hole after 4 ℃ of incubated overnight, and wash three times with PBST (pH contains 0.05%Tween-20 in 7.4 the PBS damping fluid); Each 300 μ l/ holes, 3 minutes each.
(3) sealing: sealed 300 μ l/ holes two hours with confining liquid (pH is 5% skimmed milk power, the 0.3%BSA solution of 7.4 PBS dilution) room temperature.
(4) hatching of enzyme labelled antibody and determined antigen: be diluted to corresponding concentration (0.2ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 30ng/mL, 100ng/mL, 200ng/mL) as standard items with antigen/antibody dilution (PBST) the grass carp recombinant il-10 albumen of will recombinating, (PBST does 10 to get each standard items 50 μ l and 50 μ l enzymes mark IL-10 polyclonal antibodies (HRP-IL10Ab)
3Doubly dilution) incubated at room 2 hours behind the mixing.Detection to testing sample: mix same incubated at room 2 hours with the HRP-IL10Ab of 50 μ l dilution in 1: 1000 after getting testing sample 50 μ l.Dry confining liquid after the ELISA Plate sealing is accomplished, and, add above-mentioned antigen-antibody mixed liquor, incubated at room 2 hours with 300 μ l PBST washing three times.
(5) colour developing: dry liquid in the hole, with 300 μ l PBST washing five times, each 3 minutes, the washing back is certain to be noted drying liquid in the hole, adds 100 μ l tmb substrates then, and 37 ℃ of reactions add 50 μ l 2M H after 20 minutes
2SO
4Cessation reaction.
(6) reading, result treatment: adopt ELIASA reading of data under absorbance 450nm, according to gained data computation sample inhibiting rate.Sample inhibiting rate %=(negative sample light absorption value-standard items/testing sample light absorption value)/negative sample light absorption value * 100%; Be ordinate with the standard items inhibiting rate at last; The logarithm of standard items concentration is a horizontal ordinate production standard curve, with typical curve testing sample is carried out quantitative test.
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| CN104237529A (en) * | 2014-07-10 | 2014-12-24 | 电子科技大学 | Ctenopharyngodon idellus interleukin-1beta (IL-1beta) enzyme-linked immunosorbent assay (ELISA) kit |
| CN105301258A (en) * | 2015-09-23 | 2016-02-03 | 集美大学 | Detection method of egg white content in heated foods |
| CN107505468A (en) * | 2017-08-19 | 2017-12-22 | 北京索莱宝科技有限公司 | A kind of detection reagent and its application for being used to detect Human interleukin-10 |
| CN109116036A (en) * | 2018-09-17 | 2019-01-01 | 安徽农业大学 | A kind of double-antibodies sandwich ELISA of quantitative detection grass carp interleukin-10 |
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| CN105301258A (en) * | 2015-09-23 | 2016-02-03 | 集美大学 | Detection method of egg white content in heated foods |
| CN107505468A (en) * | 2017-08-19 | 2017-12-22 | 北京索莱宝科技有限公司 | A kind of detection reagent and its application for being used to detect Human interleukin-10 |
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