RADIOIMMUNOASSAY
Dr. Micaela Cerniello, phD; Dr. Natalia Gobetto, phD.
Physiology, Faculty of Pharmacy and Biochemistry
University of Buenos Aires, UBA.
Radioimmunoassay
(RIA)
1. Overview of the radioimmunoassay
2. Procedure
3. Outcomes
1.Overview of the radioimmunoassay
Basics of RIA
➢ Competitive binding of radiolabeled antigen and unlabeled
antigen to a high affinity antibody
➢ Antigen-antibody reaction
➢ Useful to quantify very low amounts of antigen.
Antibodies
Antibodies
Antibodies
Total binding
Antibodies 100% bound
Unlabeled hormone
concentration
% Bound
Antibodies
50% bound
Antibodies
Total binding
Antibodies 100% bound
Antibodies 50% bound
50%
Cold ligand concentration (-log M)
Total Binding:
In absence of the cold antigen, all the complexes will be radioactive.
The radioactivity will be MAXIMAL.
Sample
The reduction in radioactivity in the precipitate is inversely proportional to the
amount of cold antigen in the sample.
%Bound
Total Binding
Sample
%Bound= Sample radioactivity/ Total Binding radioactivity
Do you think we need a non-especific binding tube?
To sum up…
➢ RIA is a competitive binding, between a radiolabeled antigen
and an unlabeled antigen, to a high affinity antibody
➢ The labeled antigen is mixed with the antibody at a
concentration that saturates the antigen binding sites of the
antibody
➢ As the concentration of the unlabeled antigen increases more
labeled antigen will be replaced from the binding site
RIA ADVANTAGES
➢ To quantify very low amounts of
hormones or ligands (picograms)
➢ Highly specific and sensitive
RIA DISADVANTAGES
➢ Unstable reagents
➢ Radioactive residue and waste
➢ Expensive
2. Procedure
WHAT DO WE NEED?
✓ Specific antibody against the antigen to be measured
✓ Availability of a radioactive labeled form of the antigen
✓ A method in which the antibody-bound tracer can be
separated from the unbound tracer
✓ An instrument to count radioactivity
✓ Pure antigen as standard or calibrator since a
calibration or standard curve is generated with samples
of known concentrations of the unlabeled standards.
SAMPLE PREPARATION
Samples: plasma, cell lysate, tissue
extracts, etc.
➢ Fast extraction of tissue or blood (Immediately frozen in liquid nitrogen or dry ice,
or processed immediately).
➢ Prepare samples lysates in buffer with appropriate inhibitors to prevent peptide
degradation and stop reaction.
➢ Centrifuge for 30 minutes (13000 rpm).
➢ Keep supernatant to purify peptides later.
PEPTIDE EXTRACTION
Use solid phase C18 - Sep-Pak Cartridges
extraction columns
1. Activate the columns with the proper solvents
2. Apply the sample
3. Elute with acid MeOH
4. Dry and save at -20ºC
TRACER OBTAINING
Iodination of peptides with iodine-125 (I125)
(chloramine T method)
-I125 labeled antigen are usually applied although other isotopes
such as C14 and H3 have also been used.
-Radio-labeled (I125) antigen is prepared by iodination of the pure
antigen on its tyrosine residue(s) by chloramine-T or peroxidase
methods and then separating the radio-labeled antigen from free-
isotope by gel-filtration or HPLC.
Protein
Protein
REQUIRED TUBES
Buffer Standard Samples Tracer Antibody
Tube No. (µl) (µl) (µl) (µl) (µl)
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
Incubate overnight (16 - 24 hours) at 2 - 8°C with slight agitation
SEPARATION OF THE ANTIBODY-ANTIGEN COMPLEXES
1. Add precipitating reagent (antibody + polyethylene glycol) to all tubes
and mix
2. Incubate for 30 minutes at room temperature
3. Centrifuge at 4ºC for 30 minutes at 3000 x g
4. Drop the supernatant and blot the liquid from the rims of the assay
tubes on absorbent paper mats
5. Count the radioactivity remaining in the assay tubes
CALCULATIONS
1. Calculate % Bound for standards and samples as follows:
% Bound= sample/standard radioactivity - non specific radioactivity
Total binding radioactivity - non specific radioactivity
2.Plot % bound for each standard versus the corresponding amounts of
unlabeled ligand calibrator (Standard curve)
3.Calculate the amount of peptide in the sample by interpolation in the
standard curve.
3. Outcomes
Ang-(1-7) RIA
TUBE cpm B-I %(B-I)/B0
1 4354,9 4159,5
Tube No. Buffer Standard Samples Tracer Antibody 2 4726,9 4531,5
Total 3
4
196,9
193,9
counts (B0) 1-2 100 … … 100 100 5 4471,9 4276,5 98,41
6 4486,6 4291,2 98,75
Non- 7 4231,2 4035,8 92,87
specific 8 4283,1 4087,7 94,07
9 3747,9 3552,5 81,75
binding 3-4 200 … … 100 10 3658,8 3463,4 79,70
11 3665,6 3470,2 79,86
Standards 5-26 … 100 … 100 100 12 3478,6 3283,2 75,55
13 3111,8 2916,4 67,11
Samples 27, 28, etc … … 100 100 100 14 2871,1 2675,7 61,57
15 2685,1 2489,7 57,29
16 2757,9 2562,5 58,97
17 2301,9 2106,5 48,48
18 1889,5 1694,1 38,99
19 1465,9 1270,5 29,24
20 1540,9 1345,5 30,96
21 954 758,6 17,46
22 1049,4 854 19,65
23 635,4 440 10,13
24 632,9 437,5 10,07
25 552,7 357,3 8,22
26 400,5 205,1 4,72
ANGIOTENSIN-(1-7)
Standard (dose-response) curve
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.
Standard (dose-response) curve
50%
Concentration of Ang-(1-7) in the sample
ANGIOTENSIN II
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
THANK YOU FOR
YOUR ATTENTION!!!
