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2. Procedure
3. Outcomes
1.Overview of the radioimmunoassay
Basics of RIA
➢ Antigen-antibody reaction
Antibodies
Antibodies
Total binding
Antibodies 100% bound
Unlabeled hormone
concentration
% Bound
Antibodies
50% bound
Antibodies
Total binding
Antibodies 100% bound
50%
Total Binding
Sample
➢ Unstable reagents
➢ Expensive
2. Procedure
WHAT DO WE NEED?
➢ Fast extraction of tissue or blood (Immediately frozen in liquid nitrogen or dry ice,
or processed immediately).
Protein
Protein
REQUIRED TUBES
Total Binding
(B0) 1-2 100 … … 100 100
Non-specific
binding 3-4 200 … … 100
Standards 5-26 … 100 … 100 100
Samples 27, 28, etc … … 100 100 100
4. Drop the supernatant and blot the liquid from the rims of the assay
tubes on absorbent paper mats
Concentration of Ang-(1-7) %B %B
1,000000e-013 100,000000 100,000000
9,200000e-012 93,675770 92,903350
2,300000e-011 83,553730 81,510090
4,600000e-011 82,243900 88,095010
9,200000e-011 69,209230 77,145890
1,840000e-010 63,377430 67,606450
2,300000e-010 59,495990 64,883650
4,600000e-010 45,509120 43,236460
9,200000e-010 28,850050 33,079080
1,840000e-009 20,140970 20,140970
3,680000e-009 12,165680 13,189150
7,360000e-009 3,282804 8,033215
Results obtained for the standards are used to construct a standard
(dose-response) curve from which the unknowns are calculated by
interpolation.
50%
4-Samples
1-Total tube Results obtained from the samples
2-Non-specific binding tube
ANG II (- log M) %B
3-Standard (dose-response) curve construction -10,13532 45,9453
-10,14969 46,95298
-10,07265 41,65067
Concentration of Ang II
(M) %B %B
1,000000e-013 100,000000 100,000000
2,597440e-012 109,109600 99,311900
6,493600e-012 100,564900 105,350700
1,298720e-011 88,343430 94,628740
2,597440e-011 79,511150 73,903660
5,194880e-011 53,651020 54,821820
6,493600e-011 50,816470 48,762450
1,298720e-010 29,660060 29,454660
2,597400e-010 18,876450 21,608300
5,194900e-010 11,974940 14,973810
1,038970e-009 7,045291 9,078772
6,372400e-009 4,292903 3,656157
5-Antigen concentration in the samples
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