Serum electrophoresis (SEP) Immunodiffusion ELISA

Serum protein electrophoresis (SEP)

Buffer pH 8.6

Double immunodiffusion(oucherlony technique) 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

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

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