1-20. STANDARDIZATION OF ANTIGLOBULIN SERUMS
a. Background.
(1) The most important function of antiglobulin serum is to detect RBC-
bound lgG. The present standards used by the Bureau of Biologics (BoB) are in terms
of activity against Rh antibodies only (for example, lgG). Most reagents on the market
today are prepared and standardized to detect lgG, not only in terms of Rh, but are
tested against a wide selection of lgG blood group antibodies. In addition, most of them
are prepared and standardized to detect cell-bound complement (in particular C3 and
sometimes C4). The presence of other antibodies in the reagents (for example, anti-
lgM, -lgA, -albumin, and so forth) is variable, and is usually coincidental, rather than
deliberate.
(2) The reagent can be prepared by injecting rabbits with highly purified
immunogens, or by harvesting from immuno-secreting whole human serum or protein
fractions. Injection of whole serum has several disadvantages, one being that the
animal will respond much better to single antigens, than to the many present in whole
serum; another being that antibodies to certain proteins (for example, human lgW) show
a prozone effect, whereas others (for example, antihuman beta globulin), usually do not.
Therefore, it is difficult to select one dilution that detects both lgG and complement
optimally. Because of these problems, it is more usual for the animals to be injected
with fractions of human serum, either purified proteins (for example, lgG or complement
components) or cruder fractions such as gamma and "nongamma" globulin. The
commercial houses prepare careful blends from antibodies to the different fractions, and
the antlglobulin serum is tested as below. Finally, after being licensed by the FDA, it is
distributed as antihuman globulin.
b. Specificity Testing. Following a course of immunization, the rabbits are
bled, and their serum screened for reactivity against normal non-sensitized RBCs, lgG,
and complement-sensitized RBCs. Most of the rabbit serums will contain antispecies,
which will have to be removed from the final product, by either fractionation, dilution, or
absorption with nonsensitized human RBCs, before it is sold to the consumer.
c. Antl-IgG Standardization. Antiglobulln serums are tested against
Rh-sensitized RBCs by the so-called checkerboard titration. Rh-positive RBCs are
incubated with a series of dilutions of anti-RhO(D) to yield RBCs sensitized with IgG,
varying from strongly positive to barely positive. Each sample of sensitized cells is
tested against each of a range of dilutions prepared from the rabbit anti-globulin serum.
In hyperimmunized animals, a prozone is often observed (for example, the more diluted
rabbit serum may react better than the less diluted reagent). This means that the
weakest sensitized RBCs may react with a 1:200 dilution of the antiglobulin serum, yet
not react with lower dilutions (or indeed higher dilutions). Thus, 1:200 would be the
optimal dilution of this particular reagent for the detection of Rh (lgG) antibodies. Most
commercial houses test their antiglobulin serums against many other lgG antibodies,
unfortunately, the optimal dilution for detecting lgG anti-Rh may not be the same for the
MD0846
1-25