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 freelabeled 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.
http://en.wikipedia.org/wiki/Image:ELISA-sandwich.svg
Procedure
Reagent
劑量 (ul)
Time
Then
1.Coating Ab
62 ul/11 ml PBS
100
o/n
Wash 4
次
2. Blocking
Blocking Buffer
300
1 hr
Wash 3
次
3.Standard and Sample
Reagent Diluent
100
2 hr
Wash 3
次
4.Detection Ab
62ul/11ml
Reagent
100
2 hr
Wash 3
次
5.SterptavdinHRP(1:200)
55ul/11 Reagent
100
20 min
Wash 3
次
6.Substrate
TMB
100
10 min
Wash 3
次
7.Stop
2N H2SO4
50
Competitive ELISA
http://www.geneticasysid.com.br/ELISA.htm
• 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 2,4dinitrophenol 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).
HSA
DNPBGG
DNP-HSA
DNP-BSA
BGG
BSA