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Classical Ag & Ab Reaction. Precipitation Reaction Single agar diffusion For determination of serum Immunoglobulin IgG concentration. Precipitation Reaction.
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Classical Ag & Ab Reaction Precipitation ReactionSingle agar diffusionFor determination of serum Immunoglobulin IgG concentration
Precipitation Reaction Soluble Ag(serum protein，tissue lysate or cell lysate)combine with specific Ab in appropriate condition would form visible precipitation. Precipitation Reaction can be performed in different mediums: solution single agar diffusion agar double agar diffusion
Agar diffusion test refers to soluble Ag and Ab diffuse in agar and yield white precipitation line or circle, given that they correspond to each other and with appropriate proportion. • Agar diffusion test : • Single diffusion(Radial immunodiffusion) -----refers to only diffusion of antigen or antibody, so it could be used as quantitative monitor. • Double diffusion ------refers to diffusion of both antigen and antibody, so it could be used as qualitative analysis for antigen or antibody. Agar is an amylose of large molecule. Agar could be melt at the temperature above 100℃ and concrete under 45℃ to form reticulation formation, which allows antigen and antibody diffuse freely inside it.
in the Ab-containing semisolid medium -> The area is proportional to the conc. of Ag. The region of equivalence Diagrammatic representation of radial & double immunodiffusion. precipitation reactions in gels yield visible precipitin lines; no visible precipitate forms in regions of Ab or Ag excess.
Single Agar Diffusion Test 【Purpose】Measurement of IgG concentration in serum. • IgG is the Major Ab in human serum. • It accounts for approximately 75% of the total immunoglobulin pool. • Its concentration is 1200 mg/dl. • IgG will react with Anti Human IgG antibody to form Ag-Ab complex. • In Single Radial Diffusion: one component is fixed (e.g. antigen in this case it is IgG).
【Principle】 • A melted agar solution mixed with a kind of specific Ab layer on slide. • After its solidification, punch wells and fill with known concentration of corresponding Ag which can diffuse into the agar. • If the Ag of diffusion is circular, the specific Ag-Ab complex will form visible milky white precipitation ring at the point where the ratios of Ag to Ab are in optimal proportions.
Precipitin Diameter standard curve Concentration of Ag • It’s advisable to employ a series of dilutions of a standard Ag as a reference, because the diameter of the ring shows a linear relationship to the initial concentration of the Ag. • The standard curve can be obtained and used to determine the amount of Ag in the unknown samples.
【Materials】 1.Ag: patient serum(1:50), standard serum with different concentration IgG（IgG 144U/ml，80.4μg/U）(1:18) 2. Ab: Anti-IgG antibody 3. Agar: already mixed with anti-IgG antibody 4. 1.5ml centrifuge tube, transfer pipette , slide , moist box , coordinate paper.
1:18 1:36 1:72 1:144 1:288 Standard IgG Patient serum containing anti-IgG antibody 【Methods】 a. Agar plate preparation. b. Punch wells. (diameter:3cm; space between wells: 1.2-1.5cm.) c. Add samples:5 -10μl/well d. Incubate in a humid box for 24h at 37℃.
Step 1. Dilutions of standard serum tube 1 2 3 4 N.S.(ul) 100 100 100 100 1:18 standard 100 100 100 100 Serum(ul) Dilution rate1:36 1:72 1:144 1:288 1:18 1:36 1:72 1:144 1:288 Step 2. Every 10μl standard serum is added into agar wells with different dilutions, then put agar plate in humid box. Standard IgG Patient serum anti-IgG serum 1:50 1:50
【Results】 1.Measure and record the diameter of precipitation rings. 2.Make standard curve using them as ordinate(y-axis) and dilute rate as abscissa(x-axis). 3.Measure the diameter of the precipitation ring of the tested well and check the corresponding dilute rate on the standard curve, the result of sample IgGconcentration is obtained by the formula below: Measure the IgG concentration of tested serum. Standard IgG concentration IgG(mg/m)= ×patient sera dilution rate Patient dilution rate in the curve
The standard curve of Single agar diffusion test Name: Date: Diameter （mm） 0 Dilution rate
【Attentions】 • Do not destroy the wall when add sample. • Distance among wells should be no less than 1 cm. • Bring a ruler when measure the diameter of precipitation ring.
