57
Second is the assumption that the antigen on the bead surface is labeled in the same way by
the antibody as is the antigen on the platelets. This is a weak assumption and may well be the
most difficult question in any similar quantitation attempt. The antigen on the bead is likely
~~rllAr IOI,.~ .0." .P.LA.T.E.LE.T.-_BE.A.D." .. I.X.TU.R .. E'
-
SB--f-----j
~"~
6-1----<
1
:J
36
HIE!
l!l!m
PLAt D ILUTIOIi
E!
FIll76
-"- PLATELE'T8
ALONE
... , BEADS I~ILI)"E
,'1- PLATELE'rs Iii
"IX
-:, BEADS I1~ "IX
Figure 4. An experiment using the system described in Figure 3 in which the labelling of beads alone, platelets
alone, and beads and platelets in the mixture is compared over a titration of anti-platelet antibody" The data
shows that the beads and platelets in the mixture are labelled as if they were alone.
to be oriented differently and subject to much different steric conditions than the antigen on
the cells. Thus the ratio of antibody to antigen may be very different and is difficult to
determine. In addition, beads with organic molecules exposed on their surface are very likely
to be unstable and subject to degradation over time. Therefore they frequently do not make
very good standards. The same may be said for other similar standards, such as freeze dried
or otherwise stabilized cells.
Our experience, and that of others who have attempted quantitation of antigen binding in flow
cytometers, has shown that it is possible with considerable effort, but that the degree of
uncertainty in the result seldom justifies the effort.
Second is the assumption that the antigen on the bead surface is labeled in the same way by
the antibody as is the antigen on the platelets. This is a weak assumption and may well be the
most difficult question in any similar quantitation attempt. The antigen on the bead is likely
~~rllAr IOI,.~ .0." .P.LA.T.E.LE.T.-_BE.A.D." .. I.X.TU.R .. E'
-
SB--f-----j
~"~
6-1----<
1
:J
36
HIE!
l!l!m
PLAt D ILUTIOIi
E!
FIll76
-"- PLATELE'T8
ALONE
... , BEADS I~ILI)"E
,'1- PLATELE'rs Iii
"IX
-:, BEADS I1~ "IX
Figure 4. An experiment using the system described in Figure 3 in which the labelling of beads alone, platelets
alone, and beads and platelets in the mixture is compared over a titration of anti-platelet antibody" The data
shows that the beads and platelets in the mixture are labelled as if they were alone.
to be oriented differently and subject to much different steric conditions than the antigen on
the cells. Thus the ratio of antibody to antigen may be very different and is difficult to
determine. In addition, beads with organic molecules exposed on their surface are very likely
to be unstable and subject to degradation over time. Therefore they frequently do not make
very good standards. The same may be said for other similar standards, such as freeze dried
or otherwise stabilized cells.
Our experience, and that of others who have attempted quantitation of antigen binding in flow
cytometers, has shown that it is possible with considerable effort, but that the degree of
uncertainty in the result seldom justifies the effort.