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1、ELISAELISALin ChengyuBio 042010030007Submitting Date:2012-03-21Experiment Date:2012-03-121 1IntroductionIntroduction1.11.1 Background informationBackground informationELISA(Enzyme-linked Immunosorbent Assay)is a solid-phase assay for antibodiesemploying ligands labeled with enzymes which is widely u
2、sed for immunological assays.This technique can be applied to detect antigens or antibodies for qualitative orquantitative purpose.Since enzyme reactions are very well known amplificationprocesses,the signal is generated by enzymes which are linked to the detection reagentsin fixed proportions to al
3、low accurate quantification.11.21.2 Major principlesMajor principlesFigure 1Schematic diagram of ELISA2Figure 2Procedure of indirect ELISA3As shown in Figure 1&2,the general procedure of indirect ELSIA is to:incubate theplate well with antigen,wash off unbounded antigen,incubate with 1stantibody,was
4、hoff unbounded 1st antibody,incubate with labeled 2nd antibody,wash off unbounded 2ndantibody,incubate with enzyme substrate solution,and detect optical density or otherindex showing enzyme activity.2 2Experiment OperationExperiment Operation2.12.1 Antigen coatingAntigen coating(1)Prepare an antigen
5、 solution in coating buffer(human IgG at 0.025mg/ml);(2)Pipette 200 l antigen solution to each well(Row:BG;Column:210;Column 11is negative control without antigen)of the microtiter plate;(3)Incubate the plate at 37 for 30 min;(4)Remove the antigen solution;(5)Wash each well with 200 l with PBS-T for
6、 3 times;(6)Block each well(Row:BG;Column:211)with 200 l 0.5%BSA-PBS,andincubate the plate at 37 for 30 min;(7)Remove the blocking solution;(8)Wash each well with 200 l with PBS-T for 3 times.2.22.2 Primary antibody reactionPrimary antibody reaction(1)Dilute the primary antibody(rabbit-anti-human Ig
7、G antiserum)in PBS-T fordifferent dilution(from 1:400 to 1:51,200 in 2-folds dilution);(2)Add 200 l diluted antibody solution to each well following Table 1;Table 1BCDEFG21:31:41:51:Scheme to add primary antibody61:71:81:91:10PBS-T111:4004008001,6003,2006,40012,80025,60051,200Same as Row BSame as Ro
8、w BSame as Row BSame as Row BSame as Row B(3)Incubate the plate at 37 for 1 hour;(4)Remove the primary antibody solution;(5)Wash each well with 200 l PBS-T for 3 times.2.32.3 Application of secondary antibodyApplication of secondary antibody(1)Dilute the peroxidase conjugated secondary antibody(Goat
9、-anti-rabbit IgG-HRP)inPBS-T at the dilution of 1:20,000 and 1:40,000;(2)Add 200 l secondary antibody solution to each well following Table 2;Table 2234Scheme to add secondary antibody567891011BAdd 200 l secondary antibody solution(1:20,000)to each wellCSame as Row BDSame as Row BEFAdd 200 l seconda
10、ry antibody solution(1:40,000)to each wellSame as Row EGSame as Row E(3)Incubate the plate at 37 for 1 hour;(4)Remove the secondary antibody solution;(5)Wash each well with 200 l PBS-T for 3 times.2.42.4 Substrate developmentSubstrate development(1)Add 200 l substrate solution to each well(Row:BG,Co
11、lumn:211);(2)Incubate for approximately 3 min;(3)Add 50 l 2 M H2SO4 to each well to terminate the reaction;(4)Measure optical density at 490 nm.3 3Raw data and its processingRaw data and its processing3.13.1 Raw dataRaw dataTable 3234Raw data:optical density of each well567891011B1.0790.8860.6440.49
12、80.6860.5820.5070.2600.0240.230C0.8300.7610.5920.5740.5570.5050.4760.2700.0230.248D0.7530.6570.5880.3900.6440.4850.3700.2710.0250.259EF0.5370.5100.4960.4060.4050.2770.2090.1570.0200.2590.6100.6080.5870.4810.3370.2860.1560.1510.0130.128G0.4530.5090.4540.3650.4000.2340.1430.1540.0140.1173.23.2 Data pr
13、ocessingData processingSet Row B,C,and D as Group I,and Row E,F,and G as Group II.The processed datais shown in Table 4Table 4I1:4001:8001:Processed data:optical density of each group1:1:1:1:1:No1stAb.NoAg.1,6003,2006,40012,80025,60051,2000.4950.2820.4920.1500.2700.1540.7920.7680.5900.4870.6650.0240
14、.2460.0140.122II0.5330.5100.4750.3860.402Set different dilutions of primary antibody as x axis,optical density as y axis,drawFigure 3 to illustrate their relation.Figure 3Relationship between optical density and dilutions of primary antibodyFor the reason that the curve cannot illustrate the relatio
15、nship enough,change the x axisto nature logarithm of different dilutions of primary antibody.See Figure 4:Figure 4Relationship between optical density and natural logarithm of dilutions of primary antibodyUsing linear fit for each group,we can figure out that two lines are approximatelyparallel.In t
16、he black curve in Figure 3,there is an oblivious point of inflection whichcorresponds with the dilution of 1:800.The curve after this point becomes flat,whichindicates that the binding between antigens and primary antibodies is saturated in thedilution of 1:800 and higher.This data can suggest that
17、in other immunoenzymaticexperiment,the proper dilution of primary antibody will be around,and no higher than1:800.What s more,from the red line in Figure 4 we can figure out that the optical density hasa linear relation with natural logarithm of dilutions of the primary antibody.As for comparison be
18、tween Group I and Group II,from Figure 3 we can figure out thatthe point of infection of blue curve,which corresponds with the dilution of 1:40,000,ison the left,about 1:1600.In Figure 4,the green line(1:40,000)is positionedlower than the red line(1:20,000),which is easy to understand.Lower concentr
19、ation of secondary antibody means lessbinding with primary antibody during application of secondary antibody.4 4Results and discussionResults and discussion4.14.1 ResultsResults(1)The optical density has an approximately linear relation with the natural logarithmof the dilutions of the primary antib
20、ody;(2)For secondary antibody in the dilution of1:20,000,the proper dilution of primaryantibody is 1:800;for secondary antibody in the dilution of 1:40,000,primaryantibody is recommended to be 1:1600;(3)With the same dilution of antigen and primary antibody,higher concentration ofsecondary antibody
21、will get a higher optical density;4.24.2 DiscussionDiscussion(1)(1)What is the significance of the negative control groups?What is the significance of the negative control groups?I.The no primary antibody groups proved that there is no specific bindingbetween antigen and secondary antibody,and provi
22、ded a background ofnon-specific binding between secondary antibody and antigen;The no antigen groups can provide a background of non-specific bindingbetween primary antibody and BSA.II.(2)(2)Why washing step is essential?Why washing step is essential?Washing each well with PBS-T,which contains tween
23、-20 as detergent,can wash offunbounded antigens and antibodies,including those non-specifically binding.Ifwashing step is omitted,the background index will be higher,and might causeinterference to the result.(3)(3)Why blocking step is essential?Why blocking step is essential?After the antigen coatin
24、g step,the surface of the well is not covered by antigenentirely,i.e.there is still some site leaving blank,which allows other proteins bindto them.Blocking step is to block those blank sites with non-specific bindingmaterial that will not cause interference to the experiment.Thus,the primaryantibod
25、y will only bind to the antigen coated in the first step,rather than coat on thesurface as well.(4)(4)WhatWhat s the advantage of indirect ELISA comparing with direct ELISA?s the advantage of indirect ELISA comparing with direct ELISA?I.Indirect ELISA can amplify the optical density which we measure
26、.Compared to direct ELISA,the number of secondary antibody binding tothe primary antibody is way larger than the number of primary antibodybinding to the antigen.Thus,optical density will be higher and easier tomeasure,which means a lower error;II.The secondary antibody contains HRP,which is essential for substratedevelopment.Compared to direct ELISA,indirect ELISA need only onekind of antibody contains HRP to perform many kinds of experiment,ratherthan one antibody linked to enzyme for one experiment,which isinconvenient.5 5ReferenceReference【1】http:/en.wikipedia.org/wiki/ELISA【2】http:/