ELISA 英文版实验报告.docx

Test1. Productionof antibody[principle]1. In the special humoral immune response, antigen can stimulate immune system to produce specific antibodies. Each antigen molecule has several different antigenic determinants, so it can be recognized by different B cells and then these B cells will produce different specific antibodies that can bind with epitopes specifically. The immunity serum produced by using antigen to immune animal is a mixture of many different antibodies, called polyclonal antibody. The most common method to produce polyclonal antibody is using pure antigen to immune animal. 2. The factors influencing producing of antibodies is related to antigen purity, animal, amount of antigen, ways of injection, times of immunity, etc.Primary immune animal, the antigen enter animal for the first time, the body will produce a small amount antibodies after a latency, but when second injection antigen to the animal, there will be a fast response to the antigen and a lot antibodies will be produced. So in this test, use antigen to immune mice 3 times, then we can detect antibodies in the serum.[methods]Preparation of antigen:add 3 ml N.S to tube containing SRBC and mix them.centrifuge 2000rmp, 5 min. discard the supernantrepeat above operation.Use N.S to dilute and make 20% 、40% SRBC solution.Take 0.1ml 40% to immunize the mice subcutaneously.Do the secondary and third injection after every week.Test 2.ELISA[principle]Immunological labeling techniques:Ag-Ab reactions are combined with labeling techniques. Known Ag or Ab is labeled with radioisotope, enzyme or fluorescein and unknown Ab or Ag are indirectly detected by labeled molecules. It can divided into Radioimmunoassay(RIA),Immunofluorescence technique and Enzyme Immunoassay. The Direct labeling techniques are to label each Ag and Ab, the indirect labeling techniques are to label secondary Ab.Enzyme immunoAssay (EIA): Ag-Ab reactions with enzyme-labeled Ag or Ab. Usually using horseradish peroxidase (HRP)，alkali