ELISA(酶聯(lián)免疫吸附測(cè)定)實(shí)驗(yàn)報(bào)告

1、ELISALin Che ngyu Bio 042010030007Experime nt Date: 2012-03-12Submitt ing Date: 2012-03-211 Introduction1.1 Background informationELISA (Enzyme-linked Immunosorbent Assay) is a solid-phase assay for antibodies emplo ying liga nds labeled with en zymes which is widely used for immuno logical assays.T

2、his technique can be applied to detect antigens or antibodies for qualitative or qua ntitative purpose. Si nee en zyme reacti ons are very well known amplificati on processes, the sig nal is gen erated by en zymes which are lin ked to the detect ion reage nts1in fixed proportions to allow accurate q

3、uantification.1.2 Major principlesPrnd jtlInduct ELISA 日l(shuí)ocking butTer is added bluJ trridlrnng MOteobindiig seteiNext n KJtiDlr primaryndeed0 A L11 'tle f ocorti/afy af>t)t>e)dy HfiPO I* tun a deed sHizih運(yùn) andlo tn&pm ary annbadQ TNFJ=fWi' No T113- Sflrtnnaid is convened by Hf?=C

4、:q a訊務(wù)JEEfcrmFigure 2 Procedure of indirect ELISAAs show n in Figure 1 & 2, the gen eral procedure of in direct ELSIA is to: in cubate thestplate well with antigen, wash off unbounded antigen, incubate with 1 antibody, wash off unbounded 1 st antibody, incubate with labeled 2 nd antibody, wash o

5、ff unbounded 2 nd antibody, incubate with enzyme substrate solution, and detect optical density or other in dex show ing en zyme activity.2 Experiment Operation2.1 Antigen coating(1) Prepare an antigen solution in coating buffer (human IgG at 0.025mg/ml);(2) Pipette 200 訕 antigen solution to each we

6、ll (Row: BG; Column: 210; Column 11 is n egative con trol without an tige n) of the microtiter plate;(3) Incubate the plate at 37 °C for 30 min;(4) Remove the antigen solution;(5) Wash each well with 200 d with PBS-T for 3 times;(6) Block each well (Row: BG; Column: 211) with 200 d 0.5% BSA-PBS

7、, and incubate the plate at 37 C for 30 min;(7) Remove the blocking solution;(8) Wash each well with 200 d with PBS-T for 3 times.2.2 Primary antibody reaction(1) Dilute the primary antibody (rabbit-anti-human IgG antiserum) in PBS-T for differe nt dilutio n (from 1:400 to 1:51,200 in 2-folds diluti

8、o n);(2) Add 200 d diluted antibody solution to each well following Table 1;Table 1 Scheme to add primary antibody2345678910111:1:1:1:1:1:1:1:1:B4008001,6003,2006,40012,80025,60051,200PBS-T400CSame as Row BDSame as Row BESame as Row BFSame as Row BGSame as Row B(3) Incubate the plate at 37 C for 1 h

9、our;(4) Remove the primary antibody solution;(5) Wash each well with 200 d PBS-T for 3 times.2.3 Application of secondary antibody(1) Dilute the peroxidase conjugated sec on dary an tibody (Goat-a nti-rabbit IgG-HRP) inPBS-T at the dilution of 1:20,000 and 1:40,000;(2) Add 200 d sec on dary an tibod

10、y soluti on to each well follow ing Table 2;Table 2 Scheme to add secondary antibody234567891011BAdd 200 "secondary antibody solution (1:20,000) to each wellCSame as Row BDSame as Row BEAdd 200 "secondary antibody solution (1:40,000) to each wellFSame as Row EGSame as Row E(3) Incubate the

11、 plate at 37 °C for 1 hour;(4) Remove the sec on dary an tibody solutio n;(5) Wash each well with 200 d PBS-T for 3 times.2.4 Substrate development(1) Add 200 d substrate solution to each well (Row: BG ,Column: 211);(2) Incubate for approximately 3 min;(3) Add 50 d 2 M H 2SO4 to each well to te

12、rminate the reaction;(4) Measure optical density at 490 nm.3 Raw data and its processing3.1 Raw dataTable 3 Raw data: optical density of each well234567891011B1.0790.8860.6440.4980.6860.5820.5070.2600.0240.230C0.8300.7610.5920.5740.5570.5050.4760.2700.0230.248D0.7530.6570.5880.3900.6440.4850.3700.27

13、10.0250.259E0.5370.5100.4960.4060.4050.2770.2090.1570.0200.259F0.6100.6080.5870.4810.3370.2860.1560.1510.0130.128G0.4530.5090.4540.3650.4000.2340.1430.1540.0140.1173.2 Data processingSet Row B, C, and D as Group I, and Row E, F, and G as Group II. The processed datais show n in Table 4Table 4 Proces

14、sed data: optical density of each group1:4001:8001:1,6001:3,2001:6,4001:12,8001:25,6001:51,200No1stAb.NoAg.I0.7920.7680.5900.4870.6650.4950.4920.2700.0240.246II0.5330.5100.4750.3860.4020.2820.1500.1540.0140.122Set different dilutions of primary antibody as x axis, optical density as y axis, draw Fig

15、ure 3 to illustrate their relati on.ID' J!-0.000Q310-002OM)Difkw山 diluEicih uf uruibiin diuibod$#uop 屋z-do Secondaiy aniibody I 20,000Figure 3 Relationship between optical density and dilutions of primary antibodyFor the reas on that the curve cannot illustrate the relati on ship eno ugh, cha ng

16、e the x axis to n ature logarithm of differe nt diluti ons of primary an tibody. See Figure 4:I (I - g呦dkir)antilxly I:2(LOOO ScviYndbin antihidy 1:40.0001 incjir til of worMian'翻lihcd. 1 20.0001 iTwar til m"«!ci.Tiin43n-切 1和恥 II OOO0 0 I "II191"|1|1JUO專(zhuān)-II-7-6爭(zhēng)蘭品一一口p _uux_oN

17、acum l loMihmcrdifkni diliiuonSi of primer)自 niibod、Figure 4 Relationship between optical density and natural logarithm of dilutions of primary antibodyUsing linear fit for each group, we can figure out that two lines are approximately parallel.In the black curve in Figure 3, there is an oblivious p

18、oint of inflection which corresponds with the dilution of 1:800. The curve after this point becomes flat, which indicates that the binding between antigens and primary antibodies is saturated in the dilution of 1:800 and higher. This data can suggest that in other immunoenzymatic experiment, the pro

19、per dilution of primary antibody will be around, and no higher than 1:800.What' more, from the red line in Figure 4 we can figure out that the optical den sity has a linear relation with natural logarithm of dilutions of the primary antibody.As for comparison between Group I and Group II, from F

20、igure 3 we can figure out that the point of infection of blue curve, which corresponds with the dilution of 1:40,000, is on the left, about 1:1600.In Figure 4, the green line (1:40,000) is positioned lower than the red line (1:20,000), which is easy to understand. Lower concentration of secondary an

21、tibody means less binding with primary antibody during application of secondary antibody.4 Results and discussion4.1 Results(1) The optical density has an approximately linear relation with the natural logarithm of the dilutions of the primary antibody;(2) For secondary antibody in the dilution of 1

22、:20,000, the proper dilution of primary antibody is 1:800; for secondary antibody in the dilution of 1:40,000, primary antibody is recommended to be 1:1600;(3) With the same dilution of antigen and primary antibody, higher concentration of secondary antibody will get a higher optical density;4.2 Dis

23、cussion(1) What is the significance of the negative control groups?I. The no primary antibody groups proved that there is no specific binding between antigen and secondary antibody, and provided a background of non-specific binding between secondary antibody and antigen;II. The no antigen groups can

24、 provide a background of non-specific binding between primary antibody and BSA.(2) Why washing step is essential?Washing each well with PBS-T, which contains tween-20 as detergent, can wash off unbounded antigens and antibodies, including those non-specifically binding. If washing step is omitted, t

25、he background index will be higher, and might cause interference to the result.(3) Why blocking step is essential?After the antigen coating step, the surface of the well is not covered by antigen entirely, i.e. there is still some site leaving blank, which allows other proteins bind to them. Blocking step is to block those blank sites with non-specific binding material that will not cause interference to the experiment. Thus, the prima