ELISA-英文版实验报告

Test1. Production of 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:

1.add 3 ml N.S to tube containing SRBC and mix them.

2.centrifuge 2000rmp, 5 min.

3.discard the supernant

4.repeat above operation.

e N.S to dilute and make 20% 、40% SRBC solution.

6.Take 0.1ml 40% to immunize the mice subcutaneously.

7.Do the secondary and third injection after every week.

Test 2.ELISA

[principle]

1.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.

2.Enzyme immunoAssay (EIA): Ag-Ab reactions with enzyme-labeled

Ag or Ab. Usually using horseradish peroxidase (HRP)，alkaline phosphatase (AP) to label Ag or Ab. It can be divided into ELISA and immunochemistry.

3.ELISA:Unknown Ab or Ag in blood or culture medium are detected

by enzyme-labeled Ag or Ab. It can be divided into 3 methods,

Indirect ELISA(Known Ag, enzyme-labeled secondary Ab to detect unknown Ab ),Sandwich ELISA and Competitive ELISA.

First, the known antibody or antigen is fixed on a solid carrier. Then a sample and the corresponding enzyme-linked Ab/Ag are added to bind to the Ag/Ab attached to the solid carrier. The resulting Ag-Ab complex and the excess Ab/Ag are separated by washing. Finally the substrate is added and catalysis cause a color change which can be measured.ELISA has the advantages of high sensitivity and specificity. It combines the specificity of the Ab-Ag reaction with the catalysis of enzymes.

[methods]

1.Preparation of Ag: add 3ml N.S to tube containing defibrated

SRBC and mix. Then centrifuge 2000rmp, 5 min. add 3ml N.S to the tube and mixed, then take 2 ml packed SRBC. Add 2ml DDW and shatter SRBC and dilute with coating buffer in a ratio of 1:400.

2.Coating Ag:Add 100μL of Ag to each well of ELISA plate, then

use N.S to wash for five times.

3.Prepare the serum: remove the eyeball of the SRBC-immunized

mice and collect the blood into Ep tube. Wait for about 5 minute then centrifuge at 12000rmp for 10min. take the supernant and dilute the serum 1:10.

4.Add test serum: sign and add 100μL of solution to each well, then