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Applied and Environmental Microbiology, September 2000, p. 4124-4127, Vol. 66, No. 9
0099-2240/00/$04.00+0
Murine Monoclonal Antibodies Specific for
Lipopolysaccharide of Escherichia coli O26 and
O111
Mildred
Rivera-Betancourt and
James E.
Keen*
Roman L. Hruska U.S. Meat Animal Research
Center, Agricultural Research Service, United States Department of
Agriculture, Clay Center, Nebraska 68933
Received 27 March 2000/Accepted 16 June 2000
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ABSTRACT |
Monoclonal antibody (MAb) 12F5 reacted with 35 Escherichia
coli O26 isolates and cross-reacted with 1 of 365 non-E.
coli O26 isolates. MAb 15C4 reacted with 30 E. coli
O111 strains and 8 Salmonella O35 strains (possessing
identical O antigen) but not with 362 other bacterial strains.
Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.
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TEXT |
Escherichia coli O26 and
O111 are classic enteropathogenic and emergent enterohemorrhagic
E. coli (EHEC) serotypes. Among the 13 enteropathogenic
serogroups, E. coli O26 and O111 are epidemiologically predominant and major causes of pediatric diarrhea, especially in
developing countries (22, 29). E. coli O111
caused 28% of the 50 U.S. infantile diarrhea outbreaks from 1934 to
1987 (33). Over the past 20 years, Shiga toxin-producing O26
and O111 emerged worldwide as the clinically most important non-O157 EHEC serotypes and were linked with sporadic cases and outbreaks of
human disease, including hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (8, 9, 28). Several EHEC O26- and O111-caused outbreaks have occurred in Europe and Australia, and 25% of North American HUS cases may be attributable to non-O157 EHEC
(13). In the United States, EHEC O111:NM caused a family
cluster of diarrhea and HUS (3), and EHEC O111:H8 caused a
gastroenteritis outbreak affecting more than 50 persons (1).
Pathogenic E. coli O26 and O111 are typically identified by
agglutination, using absorbed polyclonal antibodies (PAb) against lipopolysaccharide (LPS) O antigen generated by immunizing rabbits with
E. coli reference strains (22, 26). However,
absorbed anti-E. coli O26 PAb may cross-react with E. coli possessing O antigens 4, 13, 25, 32, 100, and 102 (26) and with Pseudomonas aeruginosa O12
(34). Although no E. coli O antigens cross-react with absorbed anti-E. coli O111 PAb (26), this
PAb strongly agglutinates Salmonella O35 isolates (12,
24) because of the identical chemical structure of the E. coli O111 and Salmonella O35 O antigens (4, 14,
25). Monoclonal antibodies (MAb) reactive with E. coli
O26 (15, 27) and O111 (2, 5-7, 21, 23) have been
reported but were not characterized for diagnostic sensitivity and
specificity. The public-health importance and prevalence of pathogenic
E. coli O26 and O111 in animals, humans, and foods are
probably underestimated due to the lack of available specific
serotyping reagents and because most clinical laboratories do not
routinely serotype fecal E. coli isolates (30).
We generated MAb against E. coli O26 and O111 in order to
have accurate serotyping reagents for use in planned epidemiologic
surveys of non-O157 EHEC occurrence in livestock.
MAb were produced from splenocytes of BALB/c mice immunized with
E. coli O26:H11 (ECRC DEC 10A) or O111:NM (ECRC 95.0122) whole-bacterium antigen. Immunization, hybridoma and ascites
production, and MAb screening and characterization protocols were
previously described (10, 11). MAb were isotyped with a
commercial kit (Zymed Laboratories, Inc., South San Francisco, Calif.).
One anti-O26 MAb (12F5) and one anti-O111 MAb (15C4) were generated and
characterized. MAb diagnostic sensitivity and specificity were
estimated by enzyme-linked immunosorbent assay (ELISA) reactivity with
whole-bacterium lysates from 400 gram-negative bacterial strains: 35 E. coli O26 strains, 30 E. coli O111 strains, 26 non-E. coli O26 strains reported to react with anti-O26 PAb
(E. coli O4 [n = 14], O13, O25
[n = 6], O32, O100, and O102; P. aeruginosa O12 [n = 2]), 225 other E. coli strains of various O and H serotypes, 57 Salmonella serovars (including O35 [n = 8]), and 27 other gram-negative bacterial strains. For ELISA,
bacterial-antigen-coated plates were sequentially incubated with
MAb (diluted ascites fluid), horseradish peroxidase (HRP)-conjugated antibody against mouse immunoglobulin G (IgG) plus
mouse IgM (anti-mouse IgG+IgM), and
2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] solution (ABTS
peroxidase substrate) (10). ELISA optical density was
measured at dual wavelengths of 405 and 490 nm (OD405/490);
and OD405/490 of >0.200 was considered positive. Dot box
plots (16) of MAb ELISA OD405/490 values for
bacterial-antigen subsets were generated (Prism 3.0; Graph Pad Software
Inc., San Diego, Calif.), and MAb diagnostic-sensitivity and
-specificity point estimates with exact binomial 95% confidence
intervals (CI) were calculated (Epi Info 6.0; Centers for Disease
Control and Prevention, Atlanta, Ga.). Sensitivity was defined as the
number of MAb ELISA-positive isolates per the total number of isolates tested possessing the target (O111 or O26) antigen. Specificity was
defined as the number of MAb ELISA-nonreactive isolates per the total
number of isolates tested that did not possess the target antigen.
MAb 12F5 (IgM isotype) reacted strongly by ELISA with 35 E. coli O26 isolates (sensitivity, 100%; 95% CI of 90.0 to 100) and cross-reacted with 1 of 369 non-E. coli O26 isolates
(specificity, 99.73%; 95% CI of 98.5 to 99.99) (Fig.
1). The cross-reactive strain, E. coli O4:NM (CDC3377-85), was derived from a sporadic hemorrhagic-colitis case (32) and was subsequently retyped
by the E. coli Reference Center (ECRC), Pennsylvania State
University, University Park, as O negative:NM; the original O4 antigen
was apparently lost on passage since its 1983 isolation. MAb 12F5 was
later found to cross-react with bovine E. coli O-negative:NM field isolates (J. Keen, unpublished data). While the E. coli O26 O-antigen structure is known (20), the nature
of the MAb-defined O-antigen epitope common to E. coli O26
and E. coli O-negative:NM isolates is unknown. Importantly,
MAb 12F5 did not react by ELISA with any bacteria that cross-react with
anti-O26 PAb by agglutination.
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FIG. 1.
Dot box plots of ELISA reactivities of 343 whole-bacterium lysates with anti-E. coli O26 MAb 12F5.
ELISA plates coated with whole-bacterium antigens were incubated
sequentially with MAb 12F5 (ascites fluid, 1:7,000), anti-mouse
IgG+IgM-HRP, and ABTS solution. A MAb ELISA OD405/490 of
>0.200 was considered positive. Dots represent means of duplicate OD
values. The bottom and top edges of the superimposed box plots are the
25th and 75th distribution percentiles, respectively; the central
horizontal line is the median (50th percentile), and the central
vertical lines extend from the box as far as the data extend (range).
CRX, non-E. coli O26 bacteria known to cross-react with
anti-E. coli O26 PAb. Data for MAb ELISA reactivity with 57 Salmonella isolates are not shown.
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MAb 15C4 (IgG3 isotype) reacted strongly by ELISA with 30 E. coli O111 strains (sensitivity, 100%; 95% CI of 88.4 to 100) and
8 Salmonella O35 isolates (Fig.
2). MAb interaction with
Salmonella O35 represents true antibody-antigen reactivity,
not cross-reactivity, since the E. coli O111 and
Salmonella O35 O antigens are identical (14). MAb
15C4 did not react with 317 non-E. coli O111 or 49 Salmonella non-O35 isolates (specificity, 100%; 95% CI of
99.0 to 100).
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FIG. 2.
Dot box plots of ELISA reactivities of 351 whole-bacterium lysates with anti-E. coli O111 MAb 15C4.
ELISA plates coated with whole-bacterium antigens were incubated
sequentially with MAb 15C4 (ascites fluid; 1:64,000), anti-mouse
IgG+IgM-HRP, and ABTS solution. OD values represent means of duplicate
wells. For dot box plot interpretation, see the legend for Fig. 1. Data
for MAb ELISA reactivity with 49 non-O35 Salmonella isolates
are not shown.
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(18), followed by Western immunoblotting (31)
using semipurified LPS (17, 19) and whole-bacterium
lysates from E. coli O26 and O111 and non-E. coli
O26 and O111 as antigens, confirmed MAb O-antigen specificity.
Reactive bands from gels transferred onto polyvinylidene difluoride
membranes and incubated with MAb were revealed with HRP-conjugated
rabbit anti-mouse IgG+IgM and diaminobenzidine substrate
(10). The ladder pattern characteristic of LPS was observed
on silver-stained gels and MAb-incubated membranes (Fig. 3). MAb 12F5 reacted with LPS from
E. coli O26:H11 and O4:NM CDC3377-85 but not with LPS from
five other bacterial strains. MAb 15C4 reacted only with E. coli O111 and Salmonella enterica serovar Adelaide O35 LPS. Similar immunoblots resulted when whole-bacterium lysates were
used in place of LPS (data not shown).
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FIG. 3.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and Western blots of MAb 12F5 (anti-E. coli
O26) and 15C4 (anti-E. coli O111) on purified LPS. Lanes 1, E. coli O26:H11 H311b; lanes 2, E. coli O26:H11
DEC 10A; lanes 3, P. aeruginosa O12; lanes 4, E. coli O4:NM CDC3377-85; lanes 5, E. coli O4:H5 U4-41;
lanes 6, E. coli O111:NM Stoke W; lanes 7, E. coli O111:NM ECRC 95.1022; lanes 8, Salmonella serovar
Adelaide O35 ATCC 10718. After electrophoresis, one gel was silver
stained (A). The other two gels were transferred onto polyvinylidene
difluoride membranes and incubated with cell culture medium of MAb 12F5
(B) or MAb 15C4 (C). MAb-reactive bands were revealed with rabbit
anti-mouse IgG+IgM-HRP (1:1,000) and diaminobenzidine substrate.
Molecular mass (in kilodaltons) is indicated on the left.
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In summary, we produced and characterized MAb against E. coli O26 and O111 that were sensitive and specific as assessed by ELISA against 400 target and nontarget isolates. Immunoblots against purified LPS confirmed MAb O-chain specificity. These MAb have potential use as immunodiagnostic reagents whether used alone or in
combination with other phenotypic or genotypic markers.
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ACKNOWLEDGMENTS |
We thank the following individuals for providing bacterial strains:
R. A. Wilson, ECRC, Pennsylvania State University; J. R. Johnson, University of Minnesota, St. Paul; E. G. Sowers, CDC; E. Nystrom and R. A. Schneider, National Animal Disease Center, Ames,
Iowa; K. Ferris, National Veterinary Services Laboratories, Ames, Iowa;
and J. Lam, University of Guelph, Guelph, Ontario, Canada. We also
acknowledge S. Fryda-Bradley, K. Thomas, and R. Mlejnek for technical
assistance and J. Rosch for manuscript preparation.
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FOOTNOTES |
*
Corresponding author. Mailing address: USDA, ARS, U.S.
Meat Animal Research Center, P. O. Box 166, State Spur 18D, Clay
Center, NE 68933. Phone: (402) 762-4343. Fax: (402) 762-4375. E-mail: keen{at}emailmarc.usda.gov.
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Applied and Environmental Microbiology, September 2000, p. 4124-4127, Vol. 66, No. 9
0099-2240/00/$04.00+0
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