ELISA实验报告

Experiment: ELISA (Enzyme-linked Immunosorbent Assay)Date: 2016.10.17Objectives(1) Learn ELISA principle(2) Master ELISA technology, quantitatively determine antibody and antigen.Experimental principleThe principle of ELISA is to make antigen or antibody combine to the surface of some solid phase carrier and maintain its immunocompetence, and then make the antibody or antigen to connect with some enzyme to become enzyme labeled antigen or antibody in which the immunocompetence of both antigen or antibody and enzyme is kept. In the determination, make the specimen to be tested and enzyme linked antigen react with the antigen or antibody on the surface of solid phase carrier following difference procedures. Through washing, antigen-antibody complex formed on solid phase carrier and other materials are separated. Finally, adding substrate into enzyme reaction, the substrate will be catalyzed and become colored product. The amount of product has direct relation with the amount of enzyme on the solid phase carrier, in other words, the amount of the material to be tested in the specimen. Quantitative analysis can be conducted based on the shade of color reaction. This determination has a very high sensitivity.ELISA can be divided into mainly four types: the direct method, indirect method, double antibody sandwich method and competitive inhibition method. Also, different kinds of enzyme and substrate can be used in ELISA.Double antibody method is a kind of ELISA to determine antigen in the specimen. In this method, antibody is coated on the surface of solid phase carrier. Antigen in the sample binds with antibody on the solid phase, while other materials are washed away. Then add enzyme linked antibody to bind the antigen which are bound on the solid phase. After washing, superfluous antibody is washed away. The substrate is then added to produce color and the quantitative tests are conducted.In our experiment, we adopted double antibody sandwich method to determine mouse tumor necrosis factor alpha (TNF ) with anti-mouse TNF . The enzyme and substrate we used are Avidin-HRP and TMB respectively.Reagents and equipment(1) Equipments 40-well reaction plate coated with anti-mouse TNF ; liquid transfer gun (1ml, 200l); thermotank (37).(2) Reagents Washing liquid (PBST); sealing fluid; antigen (1000pg/ml); anti-mouse TNF ; TMB; Avidin-HRP; 1M H2SO4.Method of operation(1) Empty out the solution in the plate and clean the plate with washing liquid for three times. Top up each hole and flap on absorbent paper to clean away the remaining liquid in each hole. Add 200l sealing fluid in each hole and put the plate in 37 for 30mins.(2) Wash the plate for 3 times. Add reagents in 8 holes according to the table: (number refer to the concentration of antigen added, each hole is added 100l liquid)1234567831.25(this hole is antibody-free)Washing liquid31.2562.51252505001000Put the plate in 37 for 45mins.(3) Wash the plate for 3 times, add 100l antibody in each hole, put the plate in 37 for 30mins.(4) Wash the plate for 3 times, add 100l enzyme in each hole, put the plate in 37 for 20mins.(5) Wash the plate for 5 times, add 100l substrate in each hole, put the plate in 37 for 5mins. Observe the coloration and take a picture.(6) Add 50l 1M H2SO4 in each hole, observe the color change and take a picture.Notes:(1) All reagents shall be kept in dark place at 2-8.(2) Liquid feeding shall be limited at the bottom of holes instead of wall, without spillage.(3) When cleaning the plate manually, do not spill out the liquid in the holes in case that the adjacent holes pollute each other to cause false positive.(4) The results are only valid within 30mins after the termination of test.(5) Experiment wastes shall be treated as infectious samples.Discussion(1) From the result, we can see the gradient shade of coloration, which means the shade of coloration is in proportion to the concentrate of antigen. The exact shade of coloration can be detected by spectrophotometer, and be compared with the standard curve to get the concentrate value. (2) Every time when cleaning the plate, the washing liquid must be completely removed. Questions(1) Analyze the effect of impure enzyme labeled antibody to the testing result of antigen. (Double Antibody Sandwich Method) If the impurity can not be bond with antigen, it has no affect to the result; if the impurity can be bond with antigen, it will cause the result lower than the real conce