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1、Testi. Production of antibodyprinciple1. 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 di
2、fferent 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. T
3、he 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 injec
4、tion 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. methodsPreparation of antigen:1. add 3 ml N.S to tube containing SRBC and mix them.2. centr
5、ifuge 2000rmp, 5 min.3. discard the supernant4. repeat above operation.5. Use 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.ELISAprinciple1. Immunological labeling techniques: Ag-Ab r
6、eactions 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 te
7、chniques 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 immuno
8、chemistry.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 detectunknown Ab ),Sandwich ELISA and Competitive ELISA.First, the known antibody or antigen is fixed on a
9、 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 substrateis added and catalysis cause a color change which can be measured.ELIS
10、A has the advantages of high sensitivity and specificity. It combines the specificity of the Ab-Ag reaction with the catalysis of enzymes. methods1. 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
11、 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 100 l L 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
12、. 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 100 L of solutionto each well, thenput it in a humidified box under 37 c for 45min.5. Add secondary Ab: Discard the solution in ELISA plate and wash the plate
13、 3 times. Then add 100 L of HRP-labeled secondary Ab to each well and put it in a humidified box under 37c for 30min.6. Showing color: Discard the solution in ELISA plate and wash the plate 3 times. Then add 100 l Lof substrate solution to each well. Show color in dark for10minresultthe No.1 well is
14、 blank, No.2 is negative well, No.3 is positive well, No.4 is test well. The No.3 and No.4 became yellow while No.1 and No.2 is colorless. The color of the No.4 well is deeper than the No.3. analysisThe result in No.4 well is positive becauseit become yellow,whichmeans there are antibodies in the No
15、.4 well also indicates that SRBC-immunized mice had produced antibodies. The color of test well (No.4well) is deeper than the positive control well(No.3 well), so antibodies amount in test serum is more than that in the positive control.Test3. Hypersensitivity Test of Guinea PigprinciplesHypersensit
16、ivity refers to the self tissue injury and/or biological disorder when the sensitized individual contacts the same allergen again. Hypersensitivity is the abnormal or pathologic immune response. According to the mechanisms of hypersensitivity, it could be divided into four types, type I、 H、出 and IV.
17、 Type I hypersensitivity is also called immediate type hypersensitivity or anaphylaxis. The antigen leads to hypersensitivity is called allergen. The antibody take part in anaphylaxis is mainly IgE.Allergen-induced period: In type I hypersensitivity, allergen enters body for the first time and stimu
18、lates plasma cells to produce IgE. IgE could bind to the cell surface Fce R of the mast cells or basophils.Allergen-stimulated period ： When the same allergen enters the sensitized body again, allergen could bind to the IgE on the cell surface. Then a series of reaction happen, which lead to the cel
19、l activation and biological mediators being released from these cells. These mediators lead to the attack of type I hypersensitivity.Effect period ： histamine is the main mediator that has important biological effects. The rapid release of histamine could increase vascular permeability, lead to vaso
20、dilatation, promote smooth muscle contraction and increase secretion of glands. Histamine could lead to the low bloodpressure, dyspnea, bellyache, anaphylactic shock, and even death.methods1 .Allergen-induced injection: 2 weeks before test, inject diluted egg albumin 1ml to guinea pig subcutaneously
21、.2 .Allergen-stimulated injection: Immobilize the guinea pig. Give an intracardiac injection of 1 ml diluted egg albumin. Release the guinea pig and observe its response.resultIn a few minutes after the allergen-stimulated injection,the Dyspneaconvulsion appeared.The guinea pig began to Scratch its nose. About 10 minutes the guinea pig died.