WEEK 19

Hi guys. one more week and we will be ending our SIP.

Oh well, in this post, I am going to discuss about the development of a RIA which I had been doing to develop a corticosterone RIA. There are quite a number of assays that have to be done for example, like determining the optimum antibody concentration to be used, the right dilution for sample analysis, the optimum tracer concentration and the antibody specificity. Just to take note, determining the optimum antibody concentration and tracer concentration have to be determined first before any further tests (antibody specificity test, sample dilution) can be done.

However, in this post I m going to focus in determining the antibody specificity. In other words, determining the antibody specificity is just to see if the antibody will cross react with other compounds.

Just to recall, a normal RIA procedures will require standards, antibody, tracer and of course your samples. Usually, a RIA will require a separation step. However, because our lab uses SPA, the need for separation is eliminated (recall from last post).

Antibody Specificity

To determine how specific your antibody is, we do it through a cross reactivity assay. Like normal RIA procedures, we will incubate the antibody and tracer with the blank (assay buffer), standards and the compounds that are going to be used to determine the specificity of the antibody and then incubate overnight before counting them in the scintillation counter. Since the blank contain only the labeled tracer, it should have the highest count other than the total count (total radioactivity).

Compounds to be used for corticosterone cross reactivity assay are selected from the corticosterone synthesis pathway and the concentrations used for each compound are 10, 20, 50, 100 and 1000 ng/ml.

After counting in the scintillation counter, we will get the counts of all the compounds and then calculate the percentage of B/B0 for all compounds and plot in onto a graph of %B/B0 against log concentration.
B= Binding (counts of each compound)
B0 = Total Binding (Counts from the blank)

The antibody cross reactivity is then determined by having the standard concentration at 50% binding divided by the concentration of the competitive compounds at 50% binding, expressing as a percentage. If there are no binding of the competitive compounds to the antibody at 50% binding, the antibody will be seen as not having any cross reactivity to the compounds.

Below is a graph that i had plotted from the corticosterone cross reactivity assay that i had did.


ok. from the graph, we can only see that the red and green lines crosses the 50% B/B0 marking. So that means to say that the corticosterone antibody has cross reactivity to the compounds, cortisol and progesterone other than to its own antigen of interest ie corticosterone. From the graph, the corticosterone antibody is seen to have 100% reactivty to corticosterone, 3.8% reactivity to cortisol and 0.5% reactivity to progesterone. As the rest compounds do not cross that marking, the cross reactivity cannot be calucuated. So we take it as the antibody does not react to the compounds unless at very very very high concentration. In fact, the concentrations used for the assay are already very high. Hence, for the antibody to bind to the other compounds, the concentration has to be much higher than 1000ng/ml. Therefore, we can say that the antibody is specific for corticosterone.


Hope its understandable. Enjoy your last week at your work place.

Thanks.

Xin Yi
TG02 =)