Double isotope modification to avoid washing

3.1.4 Double isotope modification to avoid washing

A major problem with the ammonium sulphate method is the need to wash the precipitates, and thus the potential to lose precipitate or for some of the complex to dissociate. A useful development introduced by Steward (1997) was the incorporation of 22 NaCl as a marker of buffer volume. Most of the supernatant can then be removed, without accurate measurement, and the amount of free radioactive antigen remaining in the precipitate estimated from the 22 Na counts to indicate the amount of buffer solution remaining with the precipitate. The technique is pos- sible as 22 Na and 125

I have substantially different radiation energy emission spectra and so can be counted separately in a two-channel γ counter.

I channel; this is determined as part of the experimental design and is allowed for during the calculations. The method is as above (Ammonium sulphate precipitation for determining antibody affinity) except for the incorpora-

There is some overlap of 22 Na counts into the 125

tion of 22 NaCl into the assay and the inclusion of tubes containing 22 NaCl alone, or 22 NaCl plus

antigen, to estimate the spillover of 22 Na counts into the 125

I channel.

MATERIALS AND EQUIPMENT These are as for the ammonium sulphate affinity determination but in addition:

22 NaCl Two-channel g spectrometer

Note: A single-channel counter may be used by resetting and counting the sample again.

82 C H A P T E R 3: Antibody interactions with antigens

METHOD

1 Set up five microfuge tubes with the same amount of 22 NaCl in each tube (about

50 000 c.p.m. in 50 µl phosphate-buffered saline (PBS) should be sufficient) to provide replicate determinations of the total sodium counts.

2 Set up the 16 assay tubes for antiserum and control serum as in the Protocol above (see p. 79). In addition to adding varying amounts of 125 I-antigen also add the same amounts of

22 NaCl in 50 µl PBS to each tube as in step 1. This takes the place of the PBS mentioned in the Protocol.

3 Mix and incubate for 1 h at room temperature.

4 Add an equal volume of saturated ammonium sulphate solution and mix immediately.

5 Incubate for 1 h at room temperature.

6 Spin the tubes at 10 000 g for 5 min.

7 Aspirate the majority of the supernatant, taking great care not to disturb the precipitate.

8 Count the radioactivity of the 22 NaCl, 22 NaCl plus antigen, and precipitate tubes in a two-channel g counter.

Calculation

For each antigen calculate the total 125

I counts added per tube (corrected for 22 Na spillover): Nx

I=I′− y

where I′ is total counts in 125

I channel including 22 Na spillover (for tubes containing 125

I antigen

I antigen plus 22 Na), x is counts in 125

plus 22 Na), N is total counts in 22 Na channel (for tubes containing 125

I channel in tubes containing 22 Na only and y is counts in 22 Na channel in tubes containing 22 Na only. Counts in 125

I channel of experimental tubes (corrected for spillover from 22 Na): nx

i = i′ − y

where i′ is counts in 125

I channel of experimental tubes including spillover from 22 Na and n is counts in 22 Na channel of experimental tubes. In the experimental tubes containing precipitate: (a) the 22 Na counts allow a calculation of the carryover of free antigen (by determining the amount of buffer carried over); and (b) the 125

I counts allow a calculation of the concentration of antigen, both free and bound to antibody. Therefore, at each of the eight antigen concentrations:

Ni − nI % antigen bound =

IN ( − n )

where terms of the equation are defined as above. Antigen bound specifically to antibody =

× total Ag

% Ag bound by antiserum % Ag bound by control serum −

3.1DETERMINATION OF ANTIBODY AFFINITY

(thus correcting for non-specific uptake by the control serum). Free antigen = total antigen − bound antigen. Calculate antibody affinity graphically as in Section 3.1.1 above.