Week 14
Lab Technique: Radioimmunoassay
It’s the 14th week already. 6 more weeks to go. Hope you guys are doing fine. =)
Last week I mentioned about faecal extraction. So now, after the extraction has been done, we can now measure the glucocorticoid level.
To measure the glucocorticoid levels in the faecal sample, radioimmunoassay (RIA) is used. Just to refresh your memory. The principle of RIA is based on the competition between unlabelled ligand and a fixed amount of radiolabelled ligand for a limit amount of antibody. The proportion of the bound labelled ligand is inversely related to the concentration of the unlabelled ligand.
An essential step in RIA is the separation of the unbound radiolabelled ligand from the Ab-Ag complexes. This is done so that the unbound radiolabelled ligand will not interfere with the counting and hence cause inaccurate result. However, this step can make the whole RIA procedure to be time consuming and can be prone to human error due to additional steps needed as compared to using SPA.
What is SPA?
SPA refers to scintillation proximity assay that makes use of SPA reagent to eliminate the separation step. The SPA reagent contains either a secondary antibody or protein A that is bound to a fluomicrosphere. The fluomicrosphere will produce light if it is bound to a radiolabelled ligand. Light will not be produced if the SPA reagent is bound onto an unlabelled ligand. Unbound radiolabelled ligand will also not produce light.
F = Fluomicrosphere with protein A or secondary Ab attached
Ab = Primary Ab
L = Unlabelled Ligand
L* = Radiolabelled ligand
To calculate the concentration of the unlabelled ligand, a machine called the scintillation counter is used. Depending on what kind of isotope used, the scintillation counter can vary. If a beta emitting isotope is used, a beta-scintillation counter will be used and if a gamma emitting isotope is used, a gamma-scintillation counter will be used. The scintillation counter will calculate the amount of radiolabelled ligand by detecing the light and then using a software, the concentration of unlabelled ligand can be determine by interpolating from a standard curve. By detecting only the light produced by bound radiolabelled ligand, this eliminate the need for the separation step.
This is a beta-scintillation counter.
(Wallac 1414 Liquid Scintillation Counter)
Picture taken from: http://www.gmi-inc.com/Genlab/Wallac%201414%20liquid%20scintillation%20counter.html
In our lab, tritium (3H) is used. And since tritium is a beta emitting isotope, a beta-scintillation counter will be used. FYI, handling of any radioisotope compound has to be done in a radioactive room. The radioactive room looks very much like a normal lab. It is just that in front of each bench, there would be a shield and we are expected to work behind the shield for safety reason.
Again, depending on whether a beta emitting radioisotope is used or a gamma emitting isotope is used, the shield could vary. Beta emitting isotopes are weakly penetrative, hence, a plastic shield is sufficient enough to block the rays. In fact, a piece of paper is already enough to block off the beta-rays. Gamma emitting isotope such as iodine 125 is more penetrative. Hence, when using 125I, we are expected to work behind a metal shield. Also, as the rays emitted can be absorbed through skin, gloves as to be wore at all times!
And of course, we will have controls to ensure that our results are valid.
As the steps involved in carrying out the RIA is quite long, I will just give a brief outline on what is done. When measuring different ligands, different set of standard concentration, antibody and tracer will be used. Hence, for illustration, I will focus on cortisol.
First we have to prepare our standards so that a standard curve can be plotted.
6 standards are prepared with concentration consisting of 188, 375, 750, 1500, 3000 and 6000 fmol/0.1ml by serial dilution starting from the standard of the highest concentration. FYI, the standard with the highest concentration is already pre-prepared by the staff in the lab.
After which, we need to prepare the SPA reagent and antibody mixture and also the 3H cortisol tracer.
Because the controls used are in serum, there is also a need for the extraction of steroids as the steroids will be bound to its binding globulin. In this case, we don’t extract the steroids like what was done on the faeces. The controls are first diluted in distilled water and then heated at 60oC for 30 minutes. The heating is sufficient to dissociate the steroids from its binding globulin.
Next, we will dilute the faecal samples 50x. After which, 100 µl of standards, PBS, faecal samples and controls will be aliqouted into another set of test tube. PBS is used to measure the total binding of the radiolabelled ligand to the SPA reagent. 200 µl of SPA reagent and antibody mixture is then added to all tubes followed by 100 µl of 3H cortisol tracer.
The test tubes are then incubated overnight (18-24h), followed by counting it in the scintillation counter.
That’s all. Hope you guys understand. I know, the post is very long. Oops. x_x
Xin Yi
TG02
posted by THE CODEC 5 @ 1:42 PM
8 Comments:
hihi, Xinyi:
there are few questions for ya...
you mentioned that the separation of the unbound radiolabelled ligand fromo the Ab-Ag complexex is time consuming and can be prone to human error,
1. why is it time consuming ? how long will it takes to do the step ? is it because it involves a lot of steps?
2. what kind of human error ?
you also mention that when using 125I, we are expected to work behind a metal shield. Also, as the rays emitted can be absorbed through skin, gloves as to be wore at all times!
3. in our lab when we handle HIV samples, we need to wear double gloves, so for your case, when using 125I do you need to wear double gloves or one gloves is enough ?
yap thanks!! =)
TINGJIE
TG02
0608495H
Hey xinyi =)
Can I know why the removal or the unbound radiolabelled ligand will be time consuming and be prone to human error?
Erm .. u know of any method(s)that is actually used to remove them?
And you said your lab employed the usage of SPA reagent to remove the unbound ligands. After adding e SPA reagent and it binds to the unbound radiolabelled ligand, how do you get rid of the complexes before proceeding on to the following steps?
Thanks.
Lyn
TGo2
Hi Xin yi,
erm you mentioned you use the scintillation counter to count, what are the principles when counting?
Thanks.
Lloyd
HI..
what is a fluomicrosphere?
thanks =)
Nur Farhana
06064834B
Hi Ting Jie,
I am not sure how long it will take to do the separation step as our lab no longer carry out this step ever since SPA is used. How many steps the separation steps involved I am also unsure. But I can check it out for you.
As to why time consuming, I have to make some changes/clarification. It should be that the need for the separation step will make the whole RIA more time consuming due to the requirement of the additional number of steps.
Hence, because of the need for the additional step, it will be time consuming. Thanks. It will make changes to it.
An example of a human error could be due to pipetting skills. Human cant be as accurate as machines. So whatever, we do there can be errors made.
As to your third question, yup, when handing 125I, there is a need to wear double gloves for double protection. One gloves will be enough if 3H is used.
Xin Yi
TG02
Hi lyn,
Your first question is quite similar to ting jie. So, I guess you can refer to the previous post.
As for why is it prone to human error is because of human intervention. So, the more human intervention there is, the more the step is prone to human error.
There are 3 methods that I know of to remove them. They are
1. Charcoal adsorption technique (traps free- labelled ligand)
2.Precipitation technique (eg. Precipitate the antigen-antibody complexes by adding a "second" antibody directed against the first)
3. Solid phase technique (eg. one reactant will be absorb to the solid surface)
About your third question, maybe I am unclear in my post. The SPA reagent does not remove unbound ligand. The use of SPA is to eliminate the separation step and not to be added to bind to unbound radiolabelled ligand for removal.
When the SPA reagent is added, it will bind to both radiolabelled ligand and unradiolabelled ligand. When a radiolabelled ligand get attached to the SPA reagent, light will be produce and vice versa if an unradiolabelled ligand get attached to the SPA reagent, no light will be produce. Unbound radiolabelled ligand not bound SPA will not produce any light.
The purpose for the light production is to allow detection by the scintillation counter for measurement. The scintillation counter will detect the light and count it, giving us the amount of radiolabelled ligand. Since unbound radiolabelled ligand will not produce any light, it will not interfere with the counting and thus affect the actual amount of radiolabelled ligand bound. Therefore, there is no need for the removal of the unbound radiolabelled ligand from the complexes.
Hope you understand
If it is still unclear, you can clear your doubts with me.
Xin Yi
TG02
Hi Lloyd,
I am not sure what are the principle of the scintillation counter. All i know for now is that it will detect the light produce from the bound radiolabelled ligand and count it. You can set the machine to count a sample for how long. The results will then appear as counts per minute. Using a software, we then can come up with the concentration.
I will find out more about the prinicple of scintillation counter and come back to you.
Xin Yi
TG02
Hi Farhana,
A fluomicrosphere is just a bead coated with a protein A or a secondary anibody when bound to a radiolabelled ligand will produce light.
Xin Yi
TG02
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