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0095-1137/82/1 10953-04$02.00/0
Copyright ©C 1982, American Society for Microbiology
Plague, caused by Yersinia pestis, is a disease bodies from rabbit anti-plague vaccine hyper-
with considerable epidemic potential in the immune serum (8) or from mouse monoclonal
western United States and many other areas of anti-fraction I ascitic fluid (J. E. Williams,
the world where wild rodents are reservoirs of manuscript in preparation) in pH 9.6 carbonate
infection. The rapid detection of new outbreaks buffer at approximately 1 ,ug of immunoglobulin
of rodent plague permits the timely application per bead (estimated by the absorbance at 280
of control measures to reduce this public health nm) and held at 4°C in the antibody-buffer mix-
threat. Since established procedures for the de- ture overnight or until needed (up to at least
tection and identification of Y. pestis may be 4 months). The rabbit antibodies were purified
time consuming, hazardous, or nonspecific (9), by ammonium sulfate precipitation and DEAE-
it is desirable to have a rapid, sensitive, relative- cellulose chromatography, and the monoclonal
ly safe alternative for the detection of Y. pestis- antibodies were purified by ammonium sulfate
specific antigen. Fluorescent microscopy has precipitation. A portion of the rabbit IgG
been used successfully to detect bound antigen preparation was labeled with 1251 by the chlora-
only and can present problems due to inherent mine-T method (4, 7) (specific activity, typically
subjectivity in evaluating test results. Precipitin 0.5 x 106 to 2.7 x 106 cpm/,Lg); the iodinated
reactions (5) and complement fixation reactions antibodies were useful for at least 6 months.
(3) of soluble Y. pestis fraction I antigen in tissue Under biohazard containment conditions, 1 to
extracts, rendered noninfectious by ether treat- 2 g of spleen plus liver was ground with sand and
ment, from animals infected with plague were about 5 volumes of phosphate-buffered saline
described about 30 years ago. We investigated (PBS). Concentrations of extracts, or dilutions
the detection of soluble fraction I antigen in thereof, were expressed as percent relative to
animal tissues by applying newer technology original tissue. Extracts containing soluble anti-
and materials, including highly purified fraction gen were rendered noninfectious by either of
IB (the major soluble protein antigen of Y. two procedures: (i) ether treatment and centrifu-
pestis), specific monoclonal antibodies, and sen- gation (3, 5) followed by filtration through a
sitive radioimmunoassay techniques. One ap- prefilter (Millipore Corp., Bedford, Mass.) and
proach was competition with radiolabeled anti- sterilizing filter (Millipore; SXGS 025 OS, 0.22
gen for limiting amounts of antibody in a p.m) or (ii) dilution (usually 20-fold) and filtration
staphylococcal radioimmune precipitin (St-RIP) of a few milliliters through a prefilter and se-
assay (8). This report describes the use of anti- quentially through two sterilizing filters (Milli-
body-coated beads and radiolabeled immuno- pore; SXGS 013 OS, 0.22 ,um). Procedure (ii),
globulin for the rapid detection of plague antigen which was more rapid than (i), also avoided the
at the nanogram level. hazards of handling ether. Each test sample in
The bead procedure was modified from that of 180 ,ul of buffer (PBS, 1% bovine serum albu-
Sarkkinen et al. (6). Major differences applicable min, 2% Tween 20, 1% fetal bovine serum
to this system were a short (2-h) initial incuba- inactivated at 56°C for 30 min, and 0.1% NaN3)
tion and the use of labeled specific antibody in a disposable glass tube (10 by 75 mm) was
rather than labeled anti-immunoglobulin G incubated with an antibody-coated bead at 37°C
(IgG). Briefly, polystyrene beads (Precision for approximately 2 h. The sample was aspirat-
Plastic Ball Co., Chicago, Ill.; 6.4-mm diameter, ed, and the bead was washed rapidly five times
specular finish) were coated with purified anti- with water, using a homemade apparatus for
953
954 NOTES J. CLIN. MICROBIOL.
extract, respectively. Other experiments (data showed '25I-labeled IgG binding at expected
not shown) indicated that although the presence levels with normal mouse extract seeded with
of an excess of normal mouse extract (100 ,ul of fraction IB and low binding with unseeded nor-
20%) had little effect on the binding of antigen in mal extract (Table 1). Mice from a vaccine test
small amounts of infected extract, an excessive series (10) that died 7 to 8 days after inoculation
amount of infected extract (20 ,ul of 20% extract with viable Y. pestis showed the presence of
containing >1,000 ng of antigen) did cause a antigen, whereas no antigen was found in survi-
reduction of bound 125I. Based on the foregoing vors sacrificed at 14 days post-inoculation (Ta-
experiments, we concluded that a sample size ble 1). The amount of detectable antigen corre-
equivalent to 0.5 to 1.0 .1l of 20% extract was lated with the number of viable Y. pestis
suitable for detecting the presence of antigen in organisms in the tissue suspensions and repre-
dead animals. sented sensitivity equivalent to <103 organisms
Four Y. pestis-positive field specimens (1-,ul in 0.15 mg of tissue (this may differ for other
samples of ether-treated extracts from prairie strains of Y. pestis or other animal species). The
dogs and ground squirrels) bound 6 to 10 times difference in detectable antigen found in the
more 125I-labeled IgG than did two Y. pestis- dead 50% lethal dose mouse, compared with the
negative specimens (Table 1). Beads coated with dead vaccinated mouse, could represent an ef-
rabbit anti-vaccine IgG bound more labeled anti- fect of vaccine-induced antibody or simply a
body with positive samples than did monoclonal random-sampling variation. The possibility that
antibody-coated beads, possibly reflecting a dif- detectable antigen was present in the survivors
ference in the efficiency of coating or binding of at some time before sacrifice cannot be ruled
antigens other than fraction I. out.
The evaluation of the filtration-only procedure Specificity was evaluated with respect to two