Practical - immunoassay

Question 1

What is immunoelectrophoresis?

Answer 1

Combination of antibody with antigen can result in the formation of a precipitate if the antigen is soluble and if antigen and antibody are present in the correct proportions. One way to detect antibody to soluble antigen is to allow the two to diffuse towards each other in a semi-solid medium such as agar gel (double diffusion in gels). At the point at which diffusing antigen meets diffusing antibody a precipitin line is formed. One way to test the purity of an antigen is to separate its components by electrophoresis in agar and then to detect these components by immunodiffusion, using an antiserum against the antigen preparation. This combination of techniques is known as immunoelectrophoresis.

Question 2

What pH is the serum?

Answer 2

At pH 8.2 serum proteins have a net negative charge and migrate towards the anode under the influence of an electric field.

Question 3

What is the pH of agar?

Answer 3

Agar has a net negative charge owing to the high concentrations of sulphate ions, with the result that there is a net flow of water towards the cathode.

Question 4

What is the antiserum used?

Answer 4

The antiserum has been produced in sheep against whole human serum and therefore contains antibodies against all the individual serum proteins.

Question 5

What is the pH of the gamma globins?

Answer 5

At pH 8.2 gamma globulins have very little charge and move only slowly towards the anode; but because of the flow of water towards the cathode they are swept back beyond the starting point and appear to have migrated towards the cathode.

Question 6

What is radical immunodiffusion?

Answer 6

This is a useful method for determining the concentration of a protein in a mixture. Antibody to the protein in question is incorporated into the agar. When antigen is placed in wells cut into the agar and allowed to diffuse out, a precipitin ring is produced. The diameter2 of this ring is proportional to the concentration of antigen. A standard curve is set up using known concentrations of antigen.

Question 7

What is the zone of equivalence?

Answer 7

The area of the precipitate where the antigen and antibody complex has formed.

Question 8

What zones are on either side of the zone of equivalence?

Answer 8

The antigen zone of excess and the antibody zone of excess.

Question 9

What are the uses of immunodiffusion?

Answer 9

-To determine the concentration of antigens and antibodies
-To compare antigens
-To compare the relative purity of antigens

Question 10

What is the radial immunodiffusion technique used for?

Answer 10

The quantification of concentrations of antigens.

Question 11

Does the antigen or antibody move during RID?

Answer 11

Only one is free to move.

Question 12

What is the process of RID?

Answer 12

1. Solution containing antibody molecules is added to molten agar.
2. This mixture is poured onto a glass slide and allowed to be set.
3. Once set wells are cut out into the agar (4-8 wells).
4. Standard antigen solution of known concentration is added to some of the wells and the antigen solution of unknown concentrations is added to the other well.
5. The slide is left for 24-72 hours and the antigens diffuse out of the wells in all directions.
6. When the antigens diffuse out and form complexes with the antibodies in the agar they form the insoluble precipitate at their most ideal concentration point (precipitation rings), this can inform us of the concentration of the unknown solution by comparing it with the known solutions.
7. The quantity of the unknown antigen concentration is determined by a standard curve.