JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1982, p. 953-956 Vol. 16. No.

5
0095-1137/82/1 10953-04$02.00/0
Copyright ©C 1982, American Society for Microbiology

Solid-Phase Radioimmunoassay for Detection of Plague

Antigen in Animal Tissue
MARJORIE E. SOERGEL,* FREDERICK L. SCHAFFER, AND HENRY F. BLANK
Naval Biosciences Laboratory, School of Public Health, University of California, Berkeley, California 94720
Received 11 January 1982/Accepted 10 June 1982

A rapid and sensitive radioimmunoassay for the detection of soluble Yersinia

pestis fraction I antigen using antibody-coated beads and radiolabeled immuno-
globulin was applied to spleen-liver extracts, rendered noninfectious by ether
treatment or filtration, from rodents dead of plague. The procedure was capable of
detecting nanogram amounts of soluble plague antigen.

Plague, caused by Yersinia pestis, is a disease
with considerable epidemic potential in the
western United States and many other areas of
the world where wild rodents are reservoirs of
infection. The rapid detection of new outbreaks
of rodent plague permits the timely application
of control measures to reduce this public health
threat. Since established procedures for the de-
tection and identification of Y. pestis may be
time consuming, hazardous, or nonspecific (9),
it is desirable to have a rapid, sensitive, relative-
ly safe alternative for the detection of Y. pestis-
specific antigen. Fluorescent microscopy has
been used successfully to detect bound antigen
only and can present problems due to inherent
subjectivity in evaluating test results. Precipitin
reactions (5) and complement fixation reactions
(3) of soluble Y. pestis fraction I antigen in tissue
extracts, rendered noninfectious by ether treat-
ment, from animals infected with plague were
described about 30 years ago. We investigated
the detection of soluble fraction I antigen in
animal tissues by applying newer technology
and materials, including highly purified fraction
IB (the major soluble protein antigen of Y.
pestis), specific monoclonal antibodies, and sen-
sitive radioimmunoassay techniques. One ap-
proach was competition with radiolabeled anti-
gen for limiting amounts of antibody in a
staphylococcal radioimmune precipitin (St-RIP)
assay (8). This report describes the use of anti-
body-coated beads and radiolabeled immuno-
globulin for the rapid detection of plague antigen
at the nanogram level.
The bead procedure was modified from that of
Sarkkinen et al. (6). Major differences applicable
to this system were a short (2-h) initial incuba-
tion and the use of labeled specific antibody
rather than labeled anti-immunoglobulin G
(IgG). Briefly, polystyrene beads (Precision
Plastic Ball Co., Chicago, Ill.; 6.4-mm diameter,
specular finish) were coated with purified anti-
953
bodies from rabbit anti-plague vaccine hyper-
immune serum (8) or from mouse monoclonal
anti-fraction I ascitic fluid (J. E. Williams,
manuscript in preparation) in pH 9.6 carbonate
buffer at approximately 1 ,ug of immunoglobulin
per bead (estimated by the absorbance at 280
nm) and held at 4°C in the antibody-buffer mix-
ture overnight or until needed (up to at least
4 months). The rabbit antibodies were purified
by ammonium sulfate precipitation and DEAE-
cellulose chromatography, and the monoclonal
antibodies were purified by ammonium sulfate
precipitation. A portion of the rabbit IgG
preparation was labeled with 1251 by the chlora-
mine-T method (4, 7) (specific activity, typically
0.5 x 106 to 2.7 x 106 cpm/,Lg); the iodinated
antibodies were useful for at least 6 months.
Under biohazard containment conditions, 1 to
2 g of spleen plus liver was ground with sand and
about 5 volumes of phosphate-buffered saline
(PBS). Concentrations of extracts, or dilutions
thereof, were expressed as percent relative to
original tissue. Extracts containing soluble anti-
gen were rendered noninfectious by either of
two procedures: (i) ether treatment and centrifu-
gation (3, 5) followed by filtration through a
prefilter (Millipore Corp., Bedford, Mass.) and
sterilizing filter (Millipore; SXGS 025 OS, 0.22
p.m) or (ii) dilution (usually 20-fold) and filtration
of a few milliliters through a prefilter and se-
quentially through two sterilizing filters (Milli-
pore; SXGS 013 OS, 0.22 ,um). Procedure (ii),
which was more rapid than (i), also avoided the
hazards of handling ether. Each test sample in
180 ,ul of buffer (PBS, 1% bovine serum albu-
min, 2% Tween 20, 1% fetal bovine serum
inactivated at 56°C for 30 min, and 0.1% NaN3)
in a disposable glass tube (10 by 75 mm) was
incubated with an antibody-coated bead at 37°C
for approximately 2 h. The sample was aspirat-
ed, and the bead was washed rapidly five times
with water, using a homemade apparatus for
954 NOTES J. CLIN. MICROBIOL.

aspiration and water dispensing that handled 10

tubes simultaneously. Approximately 40 to 60
50OrA
monoclonal
kcpm of 125I-labeled immunoglobulins in 150 pL1 beads
of buffer was added to each bead and incubated 400 1
for approximately 2 h at 37°C. After aspiration 2:,
and washing five times to remove unbound 300 -
labeled immunoglobulins, beads were trans-
ferred to clean tubes or vials for radioassay in a 2001
gamma counter.
The effect of various amounts of input 125i-
labeled IgG was tested with 2 and 50 ng of 100 I
purified fraction IB antigen (Fig. 1). The results 9
indicated that any input level within a wide 0 L * * * * J J
range might be used; however, 40 to 60 kcpm E 2000 200 20 2 0.2 0
was selected for routine use as being the minimal
input yielding a practical level of bound radioac-
tivity with 2 ng of antigen. At this input of -oc ng Frac. I B
radioactivity, the positive/negative ratio with 2 0
ng of antigen was approximately 3. Results in m B
Fig. 2 indicate that the sensitivity of the method
extended below 2 ng. In Fig. 2A, beads coated 500
with mouse monoclonal antibodies appeared to tissue-_
be slightly superior to beads coated with rabbit 400 vaccine
antivaccine IgG; however, this apparent slight
superiority was not consistently observed. Fig- Frac. IB
ure 2B shows the binding of 125I-antibody to 300
beads coated with rabbit antivaccine IgG after
exposure to serial 10-fold dilutions of purified 200
fraction IB antigen, a commercial vaccine, and a
ground squirrel tissue extract. The curves are 100
0
0
4. I 0.1 0.01 0.001
/ ,u~ ~ ~ l (equiv/.)
Vaccine or Tissue Extract
3.- FIG. 2. Radioactivity bound to beads as a function
of dilution of antigen. (A) Comparison of the detection
E
of fraction IB with rabbit IgG-coated beads and mouse
monoclonal antibody-coated beads; identical dilutions
c 2 of purified fraction IB antigen were added to reaction
-C1
0
50
mixtures which included 1 ,ul of ether-treated and
0
50ng membrane-filtered normal mouse liver and spleen ex-
mn tract. 1251I-labeled IgG input was 59 kcpm (specific
activity, ca. 470 kcpm/,ug). (B) Antigen in a tissue
I. 2ng extract (ground squirrel 166; Table 1) and a plague
vaccine (10) compared with nanogram quantities of
0 ng purified fraction IB. The amounts of vaccine and
extract are expressed as equivalents of volumes (in
microliters) of undiluted material. For fraction IB, the
15 30 60 120 240 scale is the same as in Fig. 2A. The plague vaccine
(USP, Cutter Laboratories) contained 2 x 109 formal-
Input kcpm dehyde-killed plague bacilli per ml.
FIG. 1. Binding of '25I-labeled rabbit anti-Y. pestis
vaccine IgG to beads as a function of input radioactiv-
ity. Radiolabeled antibody was added to beads that
had been coated with rabbit anti- Y. pestis vaccine IgG
and reacted with 2 or 50 ng of purified fraction IB (2, 8) generally similar; estimates based on compari-
in buffer (PBS plus 1% bovine serum albumin, 2% sons of standard and sample near the midpoint
Tween 20, 0.1% NaN3) or buffer only. Data are from of the steep portions of the curves (e.g., 250
two experiments; actual inputs were measured during cpm) indicate antigen concentrations of approxi-
incubation. mately 300 and 200 ng/pl in vaccine and tissue
VOL. 16, 1982 NOTES 955

extract, respectively. Other experiments (data
not shown) indicated that although the presence
of an excess of normal mouse extract (100 ,ul of
20%) had little effect on the binding of antigen in
small amounts of infected extract, an excessive
amount of infected extract (20 ,ul of 20% extract
containing >1,000 ng of antigen) did cause a
reduction of bound 125I. Based on the foregoing
experiments, we concluded that a sample size
equivalent to 0.5 to 1.0 .1l of 20% extract was
suitable for detecting the presence of antigen in
dead animals.
Four Y. pestis-positive field specimens (1-,ul
samples of ether-treated extracts from prairie
dogs and ground squirrels) bound 6 to 10 times
more 125I-labeled IgG than did two Y. pestis-
negative specimens (Table 1). Beads coated with
rabbit anti-vaccine IgG bound more labeled anti-
body with positive samples than did monoclonal
antibody-coated beads, possibly reflecting a dif-
ference in the efficiency of coating or binding of
antigens other than fraction I.
The evaluation of the filtration-only procedure
showed '25I-labeled IgG binding at expected
levels with normal mouse extract seeded with
fraction IB and low binding with unseeded nor-
mal extract (Table 1). Mice from a vaccine test
series (10) that died 7 to 8 days after inoculation
with viable Y. pestis showed the presence of
antigen, whereas no antigen was found in survi-
vors sacrificed at 14 days post-inoculation (Ta-
ble 1). The amount of detectable antigen corre-
lated with the number of viable Y. pestis
organisms in the tissue suspensions and repre-
sented sensitivity equivalent to <103 organisms
in 0.15 mg of tissue (this may differ for other
strains of Y. pestis or other animal species). The
difference in detectable antigen found in the
dead 50% lethal dose mouse, compared with the
dead vaccinated mouse, could represent an ef-
fect of vaccine-induced antibody or simply a
random-sampling variation. The possibility that
detectable antigen was present in the survivors
at some time before sacrifice cannot be ruled
out.
Specificity was evaluated with respect to two

TABLE 1. Detection of Y. pestis antigen in spleen-liver extracts from plague-infected rodents

1251 (cpm bound)
Specimen
Specimen prepna AnimalsbY.
prepn' Animals' pesti.s
culture' Sapl (,ul)___________
Sample (VI) Rabbit IgG Monoclonal
beads beads
Ether treated
20% Prairie dog 288 Positive 1 362 292
Prairie dog 367 Positive 1 416 287
Prairie dog 374 Negative 1 36 50
Ground squirrel 513 Positive 1 414 309
Ground squirrel 166 Positive 1 430 307
Ground squirrel 544 Negative 1 46 36
Buffer control ND 47 31
Filtered
10% Normal mouse ND 2 76
20 73
1% (seeded)' Normal mouse ND 30 (2 ng) 234
150 (10 ng) 465
0.1%e LD50 mouse, dead 1 x 104 150 273
LDso mouse, survivor <10-2 150 36
Vaccinated mouse, dead 6 x 103 150 130
Vaccinated mouse, survivor <10-2 150 32
Normal mouse ND 150 36
a Approximate concentrations are expressed as percent relative to original tissue.
b Field specimens of prairie dogs (Cynomys gunnisoni) from Saguache County (no. 288 and 367) and Pueblo
County (no. 374), Colo., and two species of ground squirrels, Spermophilus beldingi from Crook County, Ore.
(no. 513) and Spermophilus beecheyi from Monterey County (no. 166) and Plumas County (no. 544), Calif., were
collected from May to July 1981. Specimens 288 and 166 were shipped as frozen spleen-plus-liver suspensions;
the others were frozen carcasses. The mice were from a vaccine test series (10). The vaccinated mice were
immunized with a 1:180 dilution of vaccine and challenged with approximately 500 colony-forming units of Y.
pestis; the LD50 (50% lethal dose) mice received approximately 10 colony-forming units.
' Cultures of field specimen tissue suspensions are expressed as positive or negative; culture-positive
specimens were also positive by fluorescent-antibody test. The quantitative assays of mouse specimens are
expressed as colony-forming units per milligram of original tissue. After ether treatment or final filtration, all
cultures were negative. ND, Not done.
d A 10% tissue suspension was seeded with 0.67 ,ug of fraction IB per ml, diluted 10-fold, and filtered. Nominal
amounts of antigen in the samples are indicated in parentheses.
e Filtered as a 1% extract, then diluted 10-fold for the assay.
956 NOTES
other Yersinia species, Y. pseudotuberculosis
and Y. enterocolitica. Whereas cross-reactivity
with Y. pseudotubercillosis in precipitin tests (5)
and in vivo resistance to plague by Y. enterocoli-
tica infection (1) have been reported, we found
no evidence of cross-reactivity, using either
monoclonal antibody-coated or rabbit IgG-coat-
ed beads. The tests for specificity included: (i)
Y. pseudotuberculosis serotypes I and V antigen
preparations (phenolized whole organisms used
as routine diagnostic reagents) at levels of 104 or
106 organisms or clarified supernatants from 107
organisms, (ii) Y. pseudotuberclulosis serotype
III cultured in a mouse liver-spleen extract and
then filtered, and (iii) Y. enterocolitica serotype
0:1, 2, 3 and serotype 0:8 similarly cultured and
filtered. In all tests but one, the amount of 251-
labeled IgG bound after exposure to these vari-
ous preparations was less than 1.5 times the
negative controls. The exception was a marginal
ratio of 1.77 observed with a mouse extract
heavily seeded with Y. enterocolitica serotype
0:8 on mouse monoclonal beads; however, the
ratio with the same preparation on rabbit beads
was only 1.3. Further, the binding of Y. pestis
fraction lB was not blocked by 106 killed Y.
pseudotuberculosis organisms. The St-RIP test
(8) with 125I-labeled fraction IB antigen showed
no reactivity with rabbit antisera to Y. pseludotai-
berculosis serotypes I and V nor with two hu-
man sera reactive for both Y. pseudotuber(ulo-
sis and Y. enterocolitica antigens in whole cell
agglutination tests.
These results demonstrate the applicability of
solid-phase immunoassay to the rapid, specific,
and sensitive detection of soluble plague antigen
in rodents that died with Y. pestis infection. For
this purpose, quantitative precision is not re-
quired. For other applications, e.g., the assay of
soluble fraction I in killed plague vaccines (Fig.
2B), quantitation can be achieved by concurrent
testing of multiple dilutions of specimens and
standards. The technique has potential useful-
ness for the surveillance of plague infection in
animals and as a clinical diagnostic technique.
Although not tested here, it could likely be
applied to putrified specimens (3, 5), tissues
other than liver and spleen, human biopsy or
J. CLIN. MICROBIOL.
autopsy specimens, early or grossly contaminat-
ed bacterial cultures, and, perhaps, individual
infected fleas.
We express our appreciation to Daniel Eisler of this labora-
tory for providing rabbit antiplague vaccine hyperimmune
serum, James Williams of Walter Reed Army Institute of
Research, Washington, D.C., for monoclonal antibody ascitic
fluid, Thomas Quan of the Center for Disease Control, Fort
Collins, Colo., for Y. pestis-positive and -negative field rodent
specimens, and Edith Coffey and Genevieve Nygaard of the
California Department of Health, Berkeley, for Y. psetudotli-
berculosis and Y. enterocolitica reagents and seed cultures.
Support for this work was provided by the Office of Naval
Research and Bureau of Biologics, Food and Drug Adminis-
tration.
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