Biomedical Engineering Reference
In-Depth Information
If an antigen-antibody complex has to be precipitated by a sec-
ondary antibody (e.g., antigen bound by mAb precipitated by goat
anti-(mouse-IgG) antibody), this range of equivalence must be
known. For estimation of the range of equivalence the precipitin
assay is used.
A
20% polyethyleneglycol 6000 (PEG 6000) (w/v) in ddH
2
O
Solutions/Reagents
B .1N NaOH, 0.1% SDS (w/v) in ddH
2
O
PBS
Prepare a series of 1:1 or 1:2 dilutions of the first component,
e.g., rabbit immunoglobulin or serum, in PBS. Pipet 0.5 ml of each
dilution into a 2 ml tube. Add 0.5 ml of a dilution of the second
component, e.g., goat anti-(rabbit IgG) antiserum 1:100 diluted in
PBS, to each of the first dilutions. Vortex and incubate at 37
◦
C
for 1 h or at 4
◦
C overnight. Enhance precipitation by addition of
0.25 ml of Soln. A.
Spin at 8000
×
g for 20 min and wash the pellet twice with PBS.
If unlabeled components were used, dissolve the pellet with 0.1 ml
Soln. B and determine the protein content. If a radioactive labeled
compound was involved, count for radioactivity.
Plot the amount of protein and radioactivity, respectively,
against dilution. The maximum of the obtained Heidelberger
curve indicates the range of equivalence.
References
Heidelberger M, Kendall FE (1935) J Exp Med 62:697
4.8 O
UCHTERLONY
Double-Radial Immunodiffusion
4.8.1 Purification of Agar
If no agar of improved purity is present, the agar is purified by
several dialyses. Suspend 4 g of agar in 100 ml ddH
2
O; adjust pH to
7.0 with diluted hydrochloric acid or sodium hydroxide, and heat
carefully in a water bath until a clear solution is obtained. Pour
the solution onto a plate yielding a 5- to 10-mm-thick layer. After
gelation, cut into about 1-cm
2
pieces and dialyze these pieces in
a 100-fold volume of ddH
2
O for a week. Change the water daily and
add to the last portion 0.02% sodium azide. In a closed box, the
agar remains stable at 4
◦
C for month.
4.8.2 Preparation of Slides
A
1.0 - 2.0% agar or agarose (w/v), 0.02% NaN
3
in barbital buffer,
Solutions/Reagents
pH 8.4 (cf. Protocol 4.9)
B
acetic acid/ethanol/water 1:5:4 (v/v/v)
