Antiserum sensitivity

Two factors affect the sensitivity of an antiserum for use in radio-immunoassay:

• the affinity of the antiserum for the ligand. This is usually expressed as the dissociation constant (Kdiss) of the antiserum-ligand complex
• the concentration of ligand binding sites. This may be expressed in two different ways
  • As the antibody titre - the dilution of antiserum at which 50% of the tracer ligand is bound. You will determine (approximate) values for the antibody titre when you screen the antisera for their potential usefulness in radio-immunoassay.
  • As the maximum binding capacity (Bmax) in molar units; i.e. the molar concentration of binding sites in the solution.

For the equilibrium between free [F] and bound [B] ligand, the dissociation constant is defined by

Kdiss = (Bmax * [F]) / [B] = ((Bmax - [B]) * [F]) / [B]

This can be rearranged as:

[B] / [F] = (Bmax - [B]) / Kdiss

Plotting a graph of the ratio [B] / [F] against [B] gives a straight line with:

gradient = -1 / Kdiss
x intercept = Bmax

Hence, in the example shown on the right

Kdiss = 1.94 nmol /L
Bmax = 8.1 nmol /L

(This graph of the ratio [B] / [F] against [B] is known as the Scatchard plot; it was first described by Scatchard G. Annals of the New York Academy of Sciences 51: 660-72 1949.)

Incubation conditions

All solutions are prepared in, and diluted with, phosphate buffered saline (pH 7.4) containing 1 g/L bovine serum albumin (PBS+BSA). The incubations at 4C overnight are prepared as:

100 µL of oestradiol solution (a range of dilutions from 1280 to 40 fmol /mL)
100 µL tritiated oestradiol (50,000 dpm per assay)
100 µL diluted antiserum (using the dilution that you found from screening gave ~ 50% binding of the tracer)

At the end of the incubation 200 µL of charcoal suspension in PBS+BSA is added and the mixture is centrifuged.

200 µL aliquots of the supernatant (= bound ligand) are used for liquid scintillation counting