Double immunodiffusion(oucherlony technique) 沉澱線的特征與位置 抗原抗體的特異性（ identity ; non-identity ; partial identity) 相互間比例 分子大小 擴散速度 濃度

SRID Single radial immunodiffusion gel+antibody ↓ 刺個洞 ↓ 放入 antigen ↓ 觀察沉澱的範圍 擴散至抗原抗體呈現適當比例時，可以可見明顯的環狀沉澱 用以定量檢測未知標本的抗原濃度 (mg/ml 或 U/ml) 。 可檢測並定量正常人群或患者血清中 IgG 、 IgA 及 IgM 的水平

The aera of the ring is proportional to the concentration

Enzyme-Linked Immunosorbent Assay (ELISA) A type of enzyme immunoassay in which one of the reaction components is attached to the surface of a solid phase to facilitate separation of bound- and free- labeled reactants.

"indirect" ELISA The ELISA plate is prepared in the same way as the immunoassay up to step 4. In this system. The ligand is a molecule that can detect the antibody and is covalently coupled to an enzyme such as peroxidase. This binds the test antibody After free ligand is washed away (6) the bound ligand is visualized by the addition of chromogen (7) - a colorless substrate that is acted on by the enzyme portion of the ligand to produce a colored end-product. A developed plate (8) is shown in the lower panel. The amount of test antibody is measured by assessing the amount of colored end-product by optical density scanning of the plate.

Sandwich ELISA (1) Plate is coated with a capture antibody (2) sample is added, and any antigen present binds to capture antibody (3) detecting antibody is added, and binds to antigen (4) enzyme-linked secondary antibody is added, and binds to detecting antibody (5) substrate is added, and is converted by enzyme to detectable form.

Procedure Reagent 劑量 (ul) TimeThen 1.Coating Ab62 ul/11 ml PBS100o/nWash 4 次 2. BlockingBlocking Buffer3001 hrWash 3 次 3.Standard and SampleReagent Diluent1002 hrWash 3 次 4.Detection Ab62ul/11ml Reagent 1002 hrWash 3 次 5.Sterptavdin- HRP(1:200) 55ul/11 Reagent10020 minWash 3 次 6.SubstrateTMB10010 minWash 3 次 7.Stop 2N H 2 SO 4 50

Competitive ELISA

Blocking :Tween 20, BSA, Non-fat milk Enzyme : horseradish peroxidase (HRP), Alkaline phophatase (ALP) β-galactosidase Substrate: Water soluble

Homework: An animal is immunized with bovine serum albumin to which 2,4- dinitrophenol has been conjugated (DNP-BSA). Immune serum is obtained and placed in the center well of an Ouchterlony plate, with antigens in the outer wells as shown below. If BSA (bovine serum albumin) share sequence homology with HAS (human serum albumin) but not BGG (bovine gamma globulin). DNP-BSA HSA DNP-HSA BGG DNP- BGG BSA