Question

I conducted a radial diffusion assay and my unknown sample did not yield a ring, but my control samples did. Give me 3 examples of why it didn't work and how to fix it.

Answer 1

Answer.

Radial immunodiffusion (RID) is actually an immunodiffusion technique which is being used in immunology for the determination of concentration of an antigen in a sample.In this assay technique a precipitation ring is formed due the reaction between specific antigen & antibody (formation of immuno-complex).

As,your unknown sample did not yield a ring but control samples did, it can happen due to three reasons-

1. Your unknown sample & control may contain different type of antigens. The antibody in this radial diffusion test is specific for such antigen which remains in those control samples whereas it has no binding site for that antigen which is present in your unknown sample.As a result, after the spreading of unknown antigen over the antibody containing gel,immune complex is not forming due to not occurance of antigen-antibody reaction (antibody contains no specific binding site for this respective unknown antigen sample).Thus,no precipitation ring will form.But, this ring will form in case of control samples as in this case immune complex forms.

What to do to fix it-

You must use same type of antigen molecule which is specific to the used antibody, in unknown & control sample both.

2. The antibody is not be uniformly distributed over agar on the slide. It may happen,the ring is not formed as the amount of antibody molecule remains much less at that respective place where a well for unknown sample has been created.Thus,immune complex is not forming there. But near the wells of control one, amount of antibody is much which leads to the formation of the ring.

What to do to fix it-

While doing the test,you have to maintain the uniform distribution of antibody in the agar.(In this test, agar or agarose gel is used for the movement of antigen ; antibody remain mixed with it)

3. It may happen that the concentration of the unknown sample is much higher or much lower. Because to get a precipitation ring, the antibody to antigen ratio must approaches the equivalence zone. Precipitation ring will not form if this ratio get decreased (higher concentration of antigen) or increased (lower concentration of antigen). Antibody always remains constant.

What to do to fix it-

You have to maintain the particular concentration of unknown sample antigen. If the concentration is higher then make it dilute by adding water. If the concentration is lower then add more antigens to it.