a. Engvall’s estimates of the affinity constant for the labeled
liquid-phase antibody of example 1
i. Bergland’s Scatchard Plot determination of the affinity
constant
In her affidavit submitted under 37 CFR § 1.672(b) (E128), Bergland testified that she
calculated the affinity constant of the labeled antibody from the data in Example 1 using an inverse
“Scatchard Plot” analysis. The affidavit indicates she followed the procedure described in an article
by William H.C. Walker (E57). E128, p. 3.
According to the Walker article, a Scatchard Plot is a graphical technique in which data
obtained from an immunometric assay is mathematically transformed and plotted on a graph. From
the graph, certain information about the underlying antibody/antigen reaction can be determined. For
example, the technique may be used to determine the affinity constant (K) for the reaction. E57,
p.588. Walker further indicates that, the inverse Scatchard Plot is most conveniently used by plotting
the values of Lw as the x-axis values and the values of B*/Ab* as the y-axis values. E57, p.588.
2
L is the concentration of antigen and w = 1-Ab*/B*. Ab* is defined by Walker as the assay result51
2
for a sample having a large excess of ligand. E57, p. 388. B* represents the assay value for sample
corrected for background. To obtain B*, the background level is subtracted from the value obtained
from each assayed sample. E57, p. 588-89 and Table 1. In Walker’s examples, the background is
determined from a ”zero standard” --a sample which does not contain any of the antigen to be
measured. E57, p. 589, Table 1. Proper values for Ab* and B* are important in obtaining an
accurate analysis. As noted by Walker, “[a]ppropriate limits must be set for B*, zero in the absence
of ligand, Ab* in the presence of a great excess in ligand.” E57, p.588. Thus, to carry out a
Scatchard Plot analysis requires the following: (1) antigen concentration for each sample, (2) the
result of the immunometric assay for each sample (the counts per minute or absorbance), (3) the 52
background counts or absorbance, and (4) the result of the immunometric assay for a sample having
a great excess of antigen.
51
Walker exemplified the Scatchard plot by using data obtained from an immunoradiometric assay.
E57, p. 589, Tables 1 and 2. The assay used a radioactive label rather than an enzyme label as used in Engvall’s
examples. Accordingly, Walker’s examples refer to counts bound rather than absorbance.
52
Walker refers to this as the “counts bound.” E57, p. 589.
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