![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, January 1999, p. 96-100, Vol. 6, No. 1
1071-412X/99/$00.00+0
Identification of Common Lipooligosaccharide Types
in Isolates from Patients with Otitis Media by Monoclonal Antibodies
against Nontypeable Haemophilus influenzae 9274
Tomoyo
Ueyama,1
Xin-Xing
Gu,1,*
Chao-Ming
Tsai,2
Arthur B.
Karpas,3 and
David J.
Lim1
Laboratory of Immunology, National Institute
on Deafness and Other Communication Disorders,
Rockville,1
Center for Biologics
Evaluation and Research, Food and Drug Administration,
Bethesda,2 and
Laboratory of
Developmental and Molecular Immunity, National Institute of Child
Health and Human Development, Bethesda,3
Maryland
Received 19 May 1998/Returned for modification 9 September
1998/Accepted 7 October 1998
 |
ABSTRACT |
Twenty-one murine monoclonal antibodies (MAbs) were induced by
nontypeable Haemophilus influenzae (NTHi) 9274. Nineteen
MAbs were specific for the lipooligosaccharide (LOS) as determined by
enzyme-linked immunosorbent assay (ELISA) and Western blot analysis.
When the MAbs were assayed with five LOS prototype strains by ELISA,
all bound to strain 3198 LOS (type III), while six of the MAbs were
also reactive with LOSs from strain 1479 (type I), 5657 (type IV), or
7502 (type V). Ten MAbs had complement-mediated bactericidal activity,
and three MAbs were opsonophagocytic against the homologous strain.
Five LOS MAbs with different specificities were used to analyze 155 NTHi clinical isolates from the United States and from Japan. These
isolates were classified into nine groups by ELISA. Only four isolates
(2.6%) were not recognized by any of the five MAbs. Most of the
isolates (91.6%) were in four groups which bound three of the five
MAbs. One of three MAbs, 6347C11, had strong activity against the
homologous strain and was also bactericidal to 45 clinical isolates
(29%) which belonged to the four common patterns (25 belonged to
pattern 1). These data indicate that these MAbs can be used for LOS
typing in which almost all NTHi strains can be typed according to the
LOS antigenicity. Among NTHi, at least one conserved LOS epitope which
is a target of bactericidal antibodies exists. We conclude that strain
9274 LOS, which is the target for bactericidal antibodies, is a
candidate for LOS-based NTHi vaccines.
| |
INTRODUCTION |
Nontypeable Haemophilus
influenzae (NTHi) is an important cause of otitis media (OM) in
children and respiratory tract diseases in adults (12, 16,
17). NTHi accounts for 25 to 30% of acute OM and for a larger
percentage of cases of chronic OM with effusion (4, 23).
These numbers may underestimate the level because a recent study
indicated that live NTHi could be found in a large percentage of
culture-negative fluid from OM (20). Since NTHi lacks a
capsular polysaccharide, lipooligosaccharide (LOS) is believed to be a
major surface-exposed saccharide antigen and a possible virulence
factor of NTHi OM (3, 11). The LOS is also a potentially
protective antigen for NTHi infection because human antibodies showed
bactericidal activity in vitro (1), and a mouse monoclonal
antibody (MAb) enhanced opsonization and bacterial clearance in a
murine pulmonary challenge model (15). We showed that NTHi
LOS-protein conjugates elicited bactericidal antibodies in animals and
conferred protection against otitis media in chinchillas (5,
9). The LOS epitopes which elicit these biologically active
antibodies in the host have not been identified.
NTHi LOS contains an oligosaccharide linked to lipid A without an
O-specific polysaccharide (10, 19). One primary
oligosaccharide structure of LOS from NTHi strain 2019 has been
characterized, and it contains
Gal
1-4Glc1-(Hepa1-2Hepa1-3)4Hepa1-5anhydro-KDO (19). NTHi LOS is antigenically heterogeneous as
indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and immunologic methods. Campagnari et al. (2)
reported that about 50% of NTHi strains can be typed into 10 groups on the basis of the antigenic heterogeneity of LOS by rabbit
antisera. There are some anti-LOS MAbs which classified
81% of NTHi strains into eight patterns (18). MAbs are
useful in recognizing NTHi strains expressing new LOS types and in
identifying conserved and protective epitopes among clinical isolates.
In view of the importance and possibility of NTHi LOS as a vaccine
component, we generated MAbs against NTHi 9274 LOS in order to type
clinical isolates common to some or most LOS antigens. Strain 9274 was
selected for this study because its LOS does not have a terminal
lacto-N-neotetraose found in a variety of human cells
(14). In addition, this LOS was able to generate
bactericidal antibodies against homologous and heterologous strains
(9).
| |
MATERIALS AND METHODS |
Bacterial strains.
Ten strains of NTHi, including five
prototype strains (1479, 2019, 3198, 5657, and 7502) (2) and
strain 9274 (1a), were obtained from M. A. Apicella,
University of Iowa. One hundred strains from different areas of the
United States were obtained from H. Faden, State University of New York
at Buffalo, and another 55 strains were obtained from G. Mogi, Oita
Medical University, Japan. All strains were clinical isolates from
middle ear fluids or nasal secretions of patients with OM except the
prototype strains, which were from patients with chronic bronchitis.
Each strain was identified as NTHi by its sender, and this
identification was confirmed in our laboratory by bacterial morphology
and its requirement for both growth factors, nicotinamide adenine
dinucleotide (NAD) and hemin (Sigma Chemical Co., St. Louis, Mo.).
Strain 9274 was typed with anti-H. influenzae capsular
polysaccharide sera: a negative result indicated an NTHi strain.
Purification of LOS.
NTHi strains were grown in liquid
brain-heart infusion media supplemented with NAD and hemin as described
previously (7). LOSs were extracted from NTHi by hot
phenol-water and then purified by gel filtration (6).
Protein content was about 1% and nucleic acid content was less than
1% (21, 24).
Production of MAbs.
Female BALB/c mice were inoculated
intraperitoneally with about 108 CFU of strain 9274 alternating with 10 µg of its LOS at 10-day intervals. The total
number of injections was six per mouse. After resting for 3 months, the
mice were given a final intravenous dose of 108 CFU of
organisms 3 days prior to removal of their spleens. During the
immunization period, mouse sera were obtained and tested for their
antibody titer by enzyme-linked immunosorbent assay (ELISA).
Spleens were recovered from two immunized mice, and 1.0 × 108 spleen cells were combined with 0.5 × 108 nonsecreting Sp2/0-Ag14 myeloma cells. Fusions were
performed by the method of Kohler and Milstein (13) with
modification (8). Of the 1,152 original wells, 80%
contained colonies, and most colonies produced antibodies when screened
by whole-cell and LOS ELISAs. After further screening by Western
blotting, 21 wells containing one or two colonies with high reactivity
and different specificities were selected and cloned twice by limiting dilution. Selected clones were injected into the intraperitoneal space
of BALB/c mice primed with pristane.
MAb isotyping.
Determination of immunoglobulin (Ig) class
and subclass of the MAbs was accomplished with an Immuno Select ELISA
kit, which is a MAb-based isotyping system (GIBCO BRL, Bethesda, Md.).
LOS and whole-cell ELISAs.
An LOS ELISA was performed as
described previously (9). A whole-cell ELISA was performed
as follows. NTHi strains were grown on chocolate agar plates at 37°C
with 5% CO2 overnight. The bacteria were fixed with 0.37%
formaldehyde and adjusted to an optical density of 0.09 at 620 nm in
phosphate-buffered saline (PBS). Microtiter plates were coated with 100 µl of the suspension and evaporated at 37°C. Other steps were the
same as described for the LOS ELISA except that 3% bovine serum
albumin was used for blocking after coating.
ELISA inhibition test.
LOSs were used to inhibit the
reactions between MAb 6347C11 and the coating LOS antigen of strain
9274 (8). Briefly, LOSs of strains 9274, 3198, 2019, and
7502 underwent a serial twofold dilution with PBS, and then 200 µl of
each dilution or PBS was incubated with 200 µl of the appropriately
diluted MAb (A405, about 1.0) at 4°C
overnight. The following day, 100 µl of the incubated solutions in
triplicate was transferred to a microtiter plate coated with 9274 LOS,
and the subsequent reactions were performed as described for the ELISA.
The percentage of inhibition was calculated as follows: (1 
inhibitor's mean A405/control's mean
A405) × 100. All these assays were repeated,
and the variation was ±15%.
Western blot analysis.
Bacteria (10 µg), outer membrane
proteins (2 µg), or LOS (0.2 µg) was subjected to SDS-PAGE in a
15% polyacrylamide gel and then transferred onto nitrocellulose
membranes at 250 mA for 4 to 6 h (8). After blocking
with 3% bovine serum albumin in PBS for 1 h, the membranes were
incubated with MAb (about 1:100) for 3 h followed by goat
anti-mouse IgG or IgM labeled with alkaline phosphatase (1:1,000)
(Sigma) for 2 h. The membranes were developed using
5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium tablets
(Sigma). A duplicate gel was silver stained for LOS after SDS-PAGE
(22).
Bactericidal assay.
Each MAb was tested for
complement-mediated bactericidal activity (9). The highest
dilution of the MAbs causing >50% killing was considered the
bactericidal titer.
Inhibition of bactericidal activity was performed using LOSs to inhibit
the bactericidal activity of MAb 6347C11 to strain 9274. LOSs from
strain 9274, 3198, or 2019 were serially diluted 10-fold and incubated
with MAb 6347C11 (1:50 dilution) at 4°C overnight. The mixtures were
then used for the bactericidal assay.
Opsonophagocytic assay.
Human peripheral polymorphonuclear
leukocytes (PMNs) were separated from 50 ml of heparinized whole blood
from normal adults. Briefly, blood cells containing leukocytes were
sedimented by using Histopaque (Sigma), and the upper layer of
erythrocytes rich in PMNs was transferred into four 50-ml tubes. After
washing with Hanks' balanced salt solution (HBSS), erythrocytes in
each tube were lysed by adding 24 ml of sterile water. Within 1 min, PMNs were stabilized by adding 8 ml of 3.5% sodium chloride and brought to a total volume of 50 ml with HBSS. The cells were washed twice, resuspended in a minimal volume of HBSS, and counted with a hemocytometer.
Components of the assay included 0.1 ml of a log-phase bacterial
suspension (1 × 106 CFU/ml in HBSS), 0.1 ml of
complement-inactivated (preincubated at 56°C for 30 min) MAb (1:5),
0.1 ml of 20% human AB serum as a complement source (Sigma), and 0.1 ml of PMNs (3 × 106/ml in HBSS with 0.05% gelatin).
Samples were incubated at 37°C for 30 min, diluted, and plated on
chocolate agar to determine the number (CFU) of surviving bacteria.
Phagocytosis was determined to be the percentage killed which was
calculated as (1 sample's CFU/control's CFU) × 100.
| |
RESULTS |
Screening and isotyping MAbs.
Twenty-one hybridoma cell lines
were selected on the basis of their reactivity and specificity by ELISA
and Western blotting (Table 1). Two of these were IgG3 (6253F3 and
6358G3) and the others were IgM. One MAb, 6352H9, had lambda light
chains, while the others had kappa light chains. Nineteen MAbs
recognized both the purified LOS and whole cells of strain 9274, indicating that they are LOS specific while the other two MAbs, 6341F5
and 6349E8, recognized only whole cells or outer membrane proteins.
Specificity of MAbs.
The specificity of 19 MAbs against LOS
was analyzed by ELISA with LOS antigens from five NTHi prototype
strains (Table 2). All strain 9274 LOS-binding MAbs recognized 3198 LOS (type III) but not 2019 LOS (type
II). Six of these MAbs also recognized LOSs from strain 1479 (type I),
5657 (type IV), or 7502 (type V). Fifty percent of the MAbs with
different specificities were tested by Western blotting by using five
prototype LOSs and the homologous LOS 9274. Reactions by Western
blotting were consistent with those of the ELISA. Figure
1 shows a Western blot representative of
the six LOSs mentioned above with MAb 6347C11. This MAb reacted strongly with LOSs of strains 3198 and 9274, and weakly with LOS of
strain 1479.
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 1.
Silver-stained SDS-PAGE patterns (A) and Western blot
(B) of LOSs with MAb 6347C11. Lanes 1 through 8 contain 0.2 µg of
each LOS from Salmonella typhimurium Ra and Rc mutants
(lanes 1 and 2), and NTHi strains 1479, 2019, 3198, 5657, 7502, and
9274 (lanes 3 through 8). The MAb bound strongly to 3198 and 9274 LOSs
but weakly to 1479 LOS.
|
|
Bactericidal and opsonophagocytic activities of MAbs.
All MAbs
were tested for complement-mediated bactericidal activity in vitro.
Nine MAbs showed bactericidal activity against the homologous strain,
and five of them showed high bactericidal activity; however, no
correlation between their bactericidal activity and ELISA titers was
seen (Table 1). MAbs were also assayed for opsonophagocytic activity
using the homologous strain 9274. Only three showed opsonophagocytic
activity (Table 3).
Typing of clinical isolates with selected MAbs.
Five LOS MAbs
(6253F3, 6263F4, 6344C1, 6345G6, and 6347C11) were selected for the
typing of 155 NTHi clinical isolates on the basis of different LOS
antigenic determinants, isotypes, and biological activities (Tables 2
and 3). Three MAbs (IgM) showed bactericidal activity against the
homologous strain 9274. One IgG and two IgM MAbs showed
opsonophagocytic activity against strain 9274. The bactericidal or
opsonophagocytic activity of the MAbs showed no correlation with their
ELISA titers.
(i) Whole-cell ELISA.
A total of 155 NTHi clinical isolates
from the United States and Japan were typed by whole-cell ELISA using
five selected LOS MAbs, and 10 typing patterns were obtained (Table
4). Overall, 97.4% (151) of the isolates
reacted with at least one MAb, accounting for eight groups.
Thirty-seven percent of the isolates belonged to pattern 1. Patterns 1 through 4 comprised 91.6% (142) of the total isolates. It is
interesting that all isolates belonging to these four patterns reacted
with three of five MAbs (6263F4, 6344C1, and 6347C11). Isolates from
both the United States and Japan showed a similar pattern of
reactivity.
The reaction patterns of five prototype strains were also determined by
ELISA with purified LOSs or whole cells as a coating antigen (Table
5). Type III and V prototype strains
showed agreement by both ELISAs while the other three prototype strains
were different. In the LOS ELISA, the five MAbs recognized four
prototype LOSs but not type II (2019 LOS). While type III LOS was
associated with pattern 1, all of the other four LOSs were grouped in
patterns 5 to 10, which accounted for only 9% of the 155 clinical
isolates. In a whole-cell ELISA, the MAbs recognized all prototype
strains, and both 1479 (type I) and 5657 (type IV) belonged to pattern 2.
(ii) Western blot analysis.
MAb 6347C11 was selected for
Western blot analysis because of its high ELISA titer, high
bactericidal and phagocytic activities, and high positive rate of ELISA
reaction with 143 clinical isolates. A total of 139 isolates (90%)
showed a positive reaction and 16 isolates showed a negative reaction
in Western blot analysis. These results were consistent with those of
the ELISA except that four isolates were positive in whole-cell ELISA
and negative in Western blot studies.
(iii) Bactericidal assay.
MAb 6347C11 was bactericidal for 30 of 100 isolates from the United States (30%) and 15 of 55 isolates
from Japan (27%) (Fig. 2). All of these
isolates belonged to one of the four major patterns and 25 of them
(55%) belonged to pattern 1. The percentage of the bactericidal
activity of MAb 6347C11 was 51, 29, 28, and 6% in the U.S. strains and
30, 29, 45, and 11% in Japanese strains for patterns 1, 2, 3, and 4, respectively.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2.
Distribution of clinical isolates in bactericidal assay
with MAb 6347C11. The x axis represents the distribution of
clinical isolates by pattern from the United States and Japan which
could be killed by the MAb 6347C11. Patterns 1 through 10 were
identified by the five LOS typing MAbs mentioned in the text. The
y axis represents the percent of bactericidal positive
strains for each pattern from the United States or Japan calculated as
follows: numbers of bactericidal positive strains/numbers of total
strains × 100. Numbers of bactericidal positive strains are
indicated above the bars.
|
|
ELISA inhibition by NTHi LOSs.
ELISA inhibition testing to
further characterize the specificity of MAb 6347C11 was performed. The
activity of the MAb was strongly inhibited by the LOSs from the
homologous strain and strain 3198 (type III). However, it was not
inhibited significantly by LOSs from strains 2019 (type II) and 7501 (type V) at a concentration up to 1.0 mg/ml (Fig.
3).
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 3.
Inhibition of MAb 6347C11 binding to LOS of NTHi strain
9274 by four LOSs as inhibitors from NTHi strains 2019, 3198, 7501, and
9274. The percent inhibition was calculated as follows: (1 inhibitor's mean A405/control's mean
A405) × 100.
|
|
Inhibition by NTHi LOSs of bactericidal activity.
The
bactericidal activity of MAb 6347C11 was inhibited 100% by the LOS
from strains 9274 and 3198 at a concentration of 10 µg/ml but not by
LOS from strain 2019 or 7501.
| |
DISCUSSION |
Previous studies showed that NTHi LOSs are antigenically
heterogeneous. Campagnari et al. (2) established an
LOS-based serogrouping system for NTHi by using rabbit sera generated
by different NTHi strains. Only 50% of their 72 strains could be typed
into 10 groups (2). Five prototype strains representing 78%
of the 36 typed strains were used in our study as references. Patrick
et al. (18) produced four MAbs directed against LOSs by
immunizing mice with six NTHi strains and assayed LOS. A total of 69 isolates were typed into nine patterns and 19% of the isolates could
not be typed because they did not react to any of these MAbs. These
studies indicated that a more comprehensive LOS typing system is
required because of antigenic diversity among NTHi strains.
We generated 21 MAbs from hybridomas after a single fusion with
the spleens of two mice immunized with LOS and whole bacteria from NTHi
strain 9274. The resulting MAbs showed different specificities by LOS
ELISA with five prototype strains (2). All MAbs bound to
strain 3198 LOS (type III) but not strain 2019 (type II), while some
MAbs also bound to other LOSs. We examined 155 clinical isolates from
the United States and Japan by using five selected LOS-specific MAbs.
The results showed that these strains could be typed into nine groups
by whole-cell ELISA. Only four isolates (2.6%) did not react with any
of the five MAbs, but reacted with MAb 6341F5, which is directed
against whole-cell or outer membrane proteins but not to LOS. These
results indicate that almost all NTHi isolates could be typed according
to LOS by these MAbs.
The majority (91.6%) of the clinical isolates were identified as
patterns 1 through 4, with the highest percentage in pattern 1 from
both geographic sources. In addition, the isolates matching these four
patterns all reacted with three (6263F4, 6344C1, and 6347C11) of the
five LOS typing MAbs. These data suggest that patterns 1 through 4 are
common LOS types for the clinical isolates, and some of the LOS
epitopes identified by these MAbs are common to the majority of the
clinical strains.
Since about 50% of the MAbs showed complement-mediated bactericidal
activity against the homologous strain, and some of them showed
opsonophagocytosis against the same strain, further studies were
performed to identify common functional epitopes among the clinical
isolates. MAb 6347C11 with both functional activities was selected for
testing all the clinical isolates by bactericidal assay in which about
30% of the strains from either source could be killed by the MAb. In
addition, all the bactericidal isolates belonged to one of the four
major groups and 55% of them belonged to pattern 1. These results
indicate that the target of the bactericidal MAb is relatively
conserved among the clinical isolates.
In summary, the LOS MAbs are useful for typing clinical isolates, and
may be potentially useful for treatment of patients with NTHi
infections. Strain 9274 LOS, which contains common epitopes with
functional targets, is a candidate for preparing LOS-based conjugate vaccines.
| |
ACKNOWLEDGMENTS |
We are grateful to Michael A. Apicella, Howard Faden, and Goro
Mogi for providing NTHi strains.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: NIDCD, NIH, 5 Research Ct., 2A31, Rockville, MD 20850. Phone: (301) 402-2581. Fax: (301) 402-4200. E-mail: guxx{at}nidcd.nih.gov.
| |
REFERENCES |
| 1.
|
Barenkamp, S. J., and F. F. Bordor.
1990.
Development of serum bactericidal activity following nontypeable Haemophilus influenzae acute otitis media.
Pediatr. Infect. Dis. J.
9:333-339[Medline].
|
| 1a.
|
Bernstein, J. M.,
H. S. Faden,
B. G. Loos,
T. F. Murphy, and P. L. Ogra.
1992.
Recurrent otitis media with non-typable Haemophilus influenzae: the role of serum bactericidal antibody.
Int. J. Pediatr. Otorhinolaryngol.
23:1-13[Medline].
|
| 2.
|
Campagnari, A. A.,
M. R. Gupta,
K. C. Dudas,
T. F. Murphy, and M. A. Apicella.
1987.
Antigenic diversity of lipooligosaccharides of nontypable Haemophilus influenzae.
Infect. Immun.
55:882-887[Abstract/Free Full Text].
|
| 3.
|
DeMaria, T. F.,
T. Yamaguchi, and D. J. Lim.
1988.
Quantitative cytological and histological changes in the middle ear after the injection of nontypable Haemophilus influenzae endotoxin, p. 320-323.
In
D. J. Lim, C. D. Bluestone, J. O. Klein, and J. D. Nelson (ed.), Recent advances in otitis media. Decker, Philadelphia, Pa.
|
| 4.
|
Giebink, G. S.
1989.
The microbiology of otitis media.
Pediatr. Infect. Dis. J.
8:S18-S20[Medline].
|
| 5.
|
Gu, X.-X.,
J. Sun,
S. Jin,
S. J. Barenkamp,
D. J. Lim,
J. B. Robbins, and J. Battey.
1997.
Detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins confers protection against otitis media in chinchillas.
Infect. Immun.
65:4488-4493[Abstract].
|
| 6.
|
Gu, X.-X., and C.-M. Tsai.
1991.
Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media.
Anal. Biochem.
196:311-318[Medline].
|
| 7.
|
Gu, X.-X.,
C.-M. Tsai,
M. A. Apicella, and D. J. Lim.
1995.
Quantitation and biological properties of released and cell-bound lipooligosaccharide from nontypeable Haemophilus influenzae.
Infect. Immun.
63:4115-4120[Abstract].
|
| 8.
|
Gu, X.-X.,
C.-M. Tsai, and A. B. Karpas.
1992.
Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.
J. Clin. Microbiol.
30:2047-2053[Abstract/Free Full Text].
|
| 9.
|
Gu, X.-X.,
C.-M. Tsai,
T. Ueyama,
S. J. Barenkamp,
J. B. Robbins, and D. J. Lim.
1996.
Synthesis, characterization, and immunological properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins.
Infect. Immun.
64:4047-4053[Abstract].
|
| 10.
|
Helander, I. M.,
B. Lindner,
H. Brade,
K. Altmann,
A. A. Lindberg,
E. T. Rietschel, and U. Zahringer.
1988.
Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd /B+. Description of a novel deep-rough chemotype.
Eur. J. Biochem.
177:483-492[Medline].
|
| 11.
|
Iino, Y.,
R. Yuasa,
Y. Kaneko, et al.
1987.
Prognosis and endotoxin contents in middle ear effusions in cases after acute otitis media.
Acta Otolaryngol. Suppl.
435:85-89[Medline].
|
| 12.
|
Klein, J. O.,
D. W. Teele, and S. L. Pelton.
1992.
New concepts in otitis media: results of investigations of the greater Boston otitis media study group.
Adv. Pediatr.
39:127-156[Medline].
|
| 13.
|
Kohler, G., and C. Milstein.
1975.
Continuous culture of fused cells secreting antibody of predetermined specificity.
Nature (London)
256:495-497[Medline].
|
| 14.
|
Mandrell, R. E.,
R. McLaughlin,
Y. A. Kwaik,
A. Lesse,
R. Yamasaki,
B. Gibson,
S. M. Spinola, and M. A. Apicella.
1992.
Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated.
Infect. Immun.
60:1322-1328[Abstract/Free Full Text].
|
| 15.
|
McGehee, J. L.,
J. D. Radolf,
G. B. Toews, and E. J. Hansen.
1989.
Effect of primary immunization on pulmonary clearance of nontypeable Haemophilus influenzae.
Am. J. Respir. Cell. Mol. Biol.
1:201-210.
|
| 16.
|
Murphy, T. F., and M. A. Apicella.
1987.
Nontypable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection.
Rev. Infect. Dis.
9:1-15[Medline].
|
| 17.
|
Musher, D. M.,
K. R. Kubitshek,
J. Crennan, and R. E. Baughn.
1983.
Pneumonia and acute febrile tracheobronchitis due to Haemophilus influenzae.
Ann. Intern. Med.
99:344-350.
|
| 18.
|
Patrick, C. C.,
A. Kimura,
M. A. Jackson,
L. Hermanstorfer,
A. Hood,
G. H. McCracken, and E. J. Hansen.
1987.
Antigenic characterization of the oligosaccharide portion of the lipooligosaccharide of nontypeable Haemophilus influenzae.
Infect. Immun.
55:2902-2911[Abstract/Free Full Text].
|
| 19.
|
Phillips, N. J.,
M. A. Apicella,
M. Griffiss, and B. W. Gibson.
1992.
Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus influenzae.
Biochemistry
31:4515-4526[Medline].
|
| 20.
|
Rayner, M. G.,
Y. Zhang,
M. C. Gorry,
Y. Chen,
J. C. Post, and G. D. Ehrlich.
1998.
Evidence of bacterial metabolic activity in culture-negative otitis media with effusion.
JAMA
279:296-299[Abstract/Free Full Text].
|
| 21.
|
Smith, P. K.,
R. I. Krohn,
G. T. Hermanson,
A. K. Mallia,
F. H. Gartner,
M. D. Provenzano,
E. K. Fujimoto,
N. M. Goeke,
B. J. Olson, and D. C. Klenk.
1985.
Measurement of protein using bicinchoninic acid.
Anal. Biochem.
150:76-85[Medline].
|
| 22.
|
Tsai, C.-M., and C. E. Frasch.
1982.
A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels.
Anal. Biochem.
119:115-119[Medline].
|
| 23.
|
Ueyama, T.,
Y. Kurono,
K. Shirabe,
M. Takeshita, and G. Mogi.
1995.
High incidence of Haemophilus influenzae in nasopharyngeal secretions and middle ear effusions as detected by PCR.
J. Clin. Microbiol.
33:1835-1838[Abstract].
|
| 24.
|
Warburg, O., and W. Christian.
1942.
Isolation and crystallization of enolase.
Biochem. Z.
310:384-421.
|
Clinical and Diagnostic Laboratory Immunology, January 1999, p. 96-100, Vol. 6, No. 1
1071-412X/99/$00.00+0
This article has been cited by other articles:
-
Hou, Y., Gu, X.-X.
(2003). Development of Peptide Mimotopes of Lipooligosaccharide from Nontypeable Haemophilus influenzae as Vaccine Candidates. J. Immunol.
170: 4373-4379
[Abstract]
[Full Text]
-
Wu, T.-H., Gu, X.-X.
(1999). Outer Membrane Proteins as a Carrier for Detoxified Lipooligosaccharide Conjugate Vaccines for Nontypeable Haemophilus influenzae. Infect. Immun.
67: 5508-5513
[Abstract]
[Full Text]
Top
Abstract
Introduction
