248
7.
"STAINING"
will follow will be a superficial discussion of some of the problems and
techniques.
Singer and Schick (1961) conjugated ferritin to a relatively pure antibody, using either m-xylylene diisocyanate or toluene 2,4-diisocyanate,
by allowing these various substances to react in suitably buffered salt
solutions. The conjugated antibody then was purified by appropriate
centrifugation to remove uncoupled y-globulin, and by dialysis. Baxendall
et al. (1963) indicate that continuous-flow paper electrophoresis can also
be used to separate the conjugate from free ferritin. A subsequent report
by Singer and McLean (1963) indicates that toluene-2,4-diisocyanate is
the preferred coupling agent, for the two isocyanate groups in each
molecule are differentially reactive (the 4-group is about ten times as
reactive as the 2-group). Therefore, in carrying out the coupling in two
steps, the 4-isocyanate is first fastened to the ferritin molecule, and then
conditions are changed so that the 2-isocyanate group is made sufficiently
active, and couples to the antibody.
Sri Ram, Tawde, Pierce, and Midgley (1963) report that ^p'-diflurom^ra'-dinitrodiphenyl sulfone is a more reliable and better understood
conjugating agent for attaching ferritin to y-globulin. This can be done
in a one-step procedure under conditions which cause little loss of the
precipitating capacity of the antibody. This compound is readily available
from General Bio-chemicals Inc., Chagrin Falls, Ohio.
Baxendall, Perlmann, and Afzelius (1962, 1963) found that the most
effective way of building up a layer of coupled antibodies at the surface
of cells in suspension was to employ an indirect or 2-layer technique
which first deposits a layer of unlabeled specific antibody upon the cells.
This is followed by a second layer of labeled anti-y-globulin prepared in
a different species of animal. The unlabeled specific antibody then acts
as an antibody with respect to its homologous antigen in the tissue on
one side, and as an antigen with respect to the labeled anti-y-globulin
on the other. Since there are a large number of active sites on the unlabeled antibody for reaction with the marker molecule, the effective
sensitivity in locating antigen with labeled antibody is greatly increased.
This method presumably also would have an advantage in that labeled
anti-y-globulin, once it had been prepared, could be used in a number
of widely different immunological experiments since it would not in
itself be specific to the reactions of interest.
In the original experiments of Singer and Schick (1961) no effort was
made to handle material requiring sectioning. The antibody employed
7.
"STAINING"
will follow will be a superficial discussion of some of the problems and
techniques.
Singer and Schick (1961) conjugated ferritin to a relatively pure antibody, using either m-xylylene diisocyanate or toluene 2,4-diisocyanate,
by allowing these various substances to react in suitably buffered salt
solutions. The conjugated antibody then was purified by appropriate
centrifugation to remove uncoupled y-globulin, and by dialysis. Baxendall
et al. (1963) indicate that continuous-flow paper electrophoresis can also
be used to separate the conjugate from free ferritin. A subsequent report
by Singer and McLean (1963) indicates that toluene-2,4-diisocyanate is
the preferred coupling agent, for the two isocyanate groups in each
molecule are differentially reactive (the 4-group is about ten times as
reactive as the 2-group). Therefore, in carrying out the coupling in two
steps, the 4-isocyanate is first fastened to the ferritin molecule, and then
conditions are changed so that the 2-isocyanate group is made sufficiently
active, and couples to the antibody.
Sri Ram, Tawde, Pierce, and Midgley (1963) report that ^p'-diflurom^ra'-dinitrodiphenyl sulfone is a more reliable and better understood
conjugating agent for attaching ferritin to y-globulin. This can be done
in a one-step procedure under conditions which cause little loss of the
precipitating capacity of the antibody. This compound is readily available
from General Bio-chemicals Inc., Chagrin Falls, Ohio.
Baxendall, Perlmann, and Afzelius (1962, 1963) found that the most
effective way of building up a layer of coupled antibodies at the surface
of cells in suspension was to employ an indirect or 2-layer technique
which first deposits a layer of unlabeled specific antibody upon the cells.
This is followed by a second layer of labeled anti-y-globulin prepared in
a different species of animal. The unlabeled specific antibody then acts
as an antibody with respect to its homologous antigen in the tissue on
one side, and as an antigen with respect to the labeled anti-y-globulin
on the other. Since there are a large number of active sites on the unlabeled antibody for reaction with the marker molecule, the effective
sensitivity in locating antigen with labeled antibody is greatly increased.
This method presumably also would have an advantage in that labeled
anti-y-globulin, once it had been prepared, could be used in a number
of widely different immunological experiments since it would not in
itself be specific to the reactions of interest.
In the original experiments of Singer and Schick (1961) no effort was
made to handle material requiring sectioning. The antibody employed