Note: Descriptions are shown in the official language in which they were submitted.
2~7~ ~2
FIELD OF INVENTIO~
This invention relates to the use of a panel of murine
hybridoma monoclonal antibodies that are directed against
the Vi and O antigens of different common serogroups of
Salmonella. These anti-Salmonella monoclonal antibodies
can be used immediately after preparation without prior
absorption as specific factor sera for serotyping of
Salmonella.
A murine hybridoma cell line that produces monoclonal
antibodies against the outer core region of the Salmonella
lipopolysaccharide has also been developed for the
detection of Salmonella
To use such anti-Salmonella monoclonal antibodies in
immunoassays for this diverse group of intestinal
pathogens, a method has been developed that uses a
combination of tnese reagents as well as a multiple time
point assay that can detect and identify as well as
differentiate between true positive findings from non-
specific reactions that are prone to occur in immunoassays
with biological specimens.
BAC~GROUND ART
Salmonella comprises a diverse group of intestinal
pathogens affecting both animals and man. Some serot~pes
of Salmonella can cause invasivP systemic diseases such as
enteric fever in man and fowl typhoid in chickens. Others
are better known to be the most common cause of food-
poisoning. Definite diagnosis of salmonellosis depends on
the detection and isolation of the organism which is then
identified by biochemical and serological means.
Serological identification of Salmonella is normally
effected by slide and/or tube agglutination technique using
polyclonal antisera prepared in experimental animals, e.g.
rabbit. Monospecific antisera for serotyping of Salmonella
are prepared by exhaustive absorption with members that
2~78~ 62
share common antigenic factors. Despite the elaborate
absorption procedures, which are timing consuming and have
inherent problem of batch to batch variation that is
difficult to control, the resulting antiserum is not truly
mono-specific and still contains a mixture of antibodies.
Thus serotyping of Salmonella based on polyclonal antisera
suffers ~rom the ill-defined nature of antisera, and there
is a need to develop a more efficient approach to raise
anti-Salmonella antibodies for serotyping.
The conventional approach for detection of Salmonella
involves culture of the organism from specimens using
standard bacteriological techniques which may involve
inoculation of the specimens into a variety of media
designed to enhance even small numbers of Sàlmonella to be
detected. One commonly employed approach is to enrich the
Salmonella in a broth medium followed by plating the
incubated broth culture onto a solid agar medium which
would allow any suspected Salmonella cells to grow up as
visible colonies. The latter are then identified by a
battery of biochemical and serological tests (Ewing W.H.
1986. Identification of Enterobacteriaceae, 4th edition.
New York: Elsevier). Because this elaborate protocol is
both time consuming (taking as long as several days) and
labour intensive (demanding a lot of experience from the
technical personnel to recognize any suspected colonies
from the final culture step on agar plate), there has
always been a need to develop more rapid and less demanding
method ~or the detection of Salmonella.
Hitherto, a number of alternative methods have been
described, such as a DNA probe method (Fitts, R., Diamond,
M., Hamilton, C., and Neri, M. 1983. DNA-DNA hybridization
assay for detection of Salmonella spp. in foods. Applied
and Environmental Microbiology, vol. 46: pp. 1146-1151;
Scholl, D.R., Kaufmann, C., Jollick, J.D., York, C.K.,
Goodrum G.R., and Charache, P. 1990. Clinicial Application
~7~2
of novel sample processing technology for the
identification of Salmonellae by using DN~ probesO Journal
of Clinical Microbiology, vol. 28: pp. 237-241),
electrioal conductance (Smith, P.J., Boardman A., and
Shutt, P.C. 1989. Detection of salmonellas in animal feeds
by electrical conductance. Journal of Applied
Bacteriology, vol. 67: pp. 575-588), and immunosensor
(Luong, J.H.T., Prusak-Sochaczewski, E., and Guilbault,
G.G. 1990. Development of a piezoimmunosensor for the
detection of Salmonella tY~himurium. Annals of the New
York Academy of Sciences, vol. 613: pp. 439-443). However
these alterative approaches have not gained wide acceptance
into the routine laboratory either because of problems in
sensitivity or because of lack of specificity.
Since one of the key tests in the conventional
approach for the identification of Salmonella rests on the
serological typing based on the use of specific antiserum
against Salmonella antigens, many alternative methods for
Salmonella detection have been developed using
~0 immunological approaches with antisera developed for the
serolo~ical typing of the bacteria (Sperber, W.H., and
Deibel, R.H. 1969. Accelerated procedure for Salmonella
detection in dried foods and feeds involving only broth
cultures and serological reactions. Applied Microbiology,
vol. 17: pp. 533-539; Gibbs, P.A., Patterson, J.T., and
Murray, J.G. 1972. The fluorescent an~ibody technique for
the detection of Salmonella in routine use. Journal of
~pplied Bacteriology, vol. 35: pp. 405-413; Minnich, S.A.,
Hartman, P.A., and Heimsch, R.C. 1982. Enzyme immunoassay
for detection of Salmonellae in foods. Applied and
Environmental Microbiology, vol. ~3: pp. 877-883;
Cerqueira-Campos, M.-L., Peterkin, P.I., and Sharpe, A.N.
1986. Improved immunological membrane filter method for
detection of food~borne Salmonella strains. Applied and
Environmental Microbiology, vol. 52: pp. 124-127; Hassan,
2~7~2
J.O., Mockett, A.P.A., Mcleod, S., and Barrow, P.A. l9gl.
Indirect antigen-trap ELISAs using polyclonal antisera for
detection of group B and D Salmonellas in chickens. Avian
Pathology, vol. 20: pp. 271-281). Indeed over the years,
there have been many immunoassays described for the
detection of Salmonella (reviewed in Tsang, R.S.W. and
Nielsen, K.H. 1992. Immunoassays for Salmonella. The
Genetic Engineer and Biotechnologist, vol. 12: pp. 14-18~,
but many still lack the specificity required due to the
numerous common cross-reactive antigens present in the
different members of the Family of Enterobacteriaceae and
the inherent properties of polyclonal hyperimmunized
e~perimental animal sera.
In the past decade, monoclonal antibodies against the
common epitopes of Salmonella flagella antigen have also
been applied in immunoassays for Salmonella (Robison, B.J.,
Pretzman, C.I., and Mattingly, J.A. 1983. Enzyme
immunoassay in which a myeloma protein is used for
detection of Salmonella. Applied and Environmental
Microbiology, vol. 45: pp. 1816-1821; Mattingly, J.A.,
Robison, B.J., Boehm, A., and Gehle, W.D. 1985. Use of
monoclonal antibodies for the detection of Salmon~lla in
foods. Journal of Food Technology, vol. 39: pp. 90-94).
However not all salmonellae are detected by such monoclonal
antibodies. Also false positive results due to cross-
reactions with Citrobacter have been reported and results
were obtained only after three days (Clayden, J.A., Alcock,
S.J., and Stringer, M.F. 1987. Enzyme linked immunosorbent
assays for the detection of Salmonella in Foods.
Immunological Techniques in Microbiology, pp. 217-229).
Therefore there is still a demand for a truly monospecific
antiserum or antibody against the genus Salmonella that
does not show cross reactions with non-Salmonella bacteria.
As mentioned above, although a number of alternative
procedures for salmonellae detection have been proposed,
~137~2
the most commonly described alternative method is
immunoassay. This is probably a re~lection o~ the
practicality of such an approach in terms of both
availability of reagents and familiarity of immunoassay
technology to laboratory personnel. However like many
other assays, the quality of the sample and the reagents
employed primarily determine the accuracy of the assay.
Presence of no~ious substances in the sample may cause non-
specific stickiness of reagents used in immunoassay leading
to high background or false positive results. Although
controls, such as inclusion of no-capture antibody or no-
enzyme detector antibody molecule, may allow the detection
of a non-specific reaction or high background, they do not
necessarily provide an answer to differentiate between true
positive results from non-specific reactions.
To develop an improved immunoassay for detection of
Salmonella, there is the requirement of a good specific
antiserum or antibody against the many di~ferent serotypes
of Salmonella, as well as an immunoassay method that would
minimize interferences from noxious substances which may be
present in the many different types o~ possible samples
that are known to or may contain Salmonella bacteria.
BROAD DESCRIPTION OF THE I~3VENTIOld
An object of the invention is to conduct serotyping of
Salmonella by employing monoclonal antibodies which can be
used without prior absorption as factor specific sera.
The existing serotyping sera are produced in
~xperimental animals by injection with Salmonella whole
cells and absorptions with related bacteria are undertaken
on the resulting antiserum to remove cross-reacting
antibodies and hence rendering the antisera monospeci~ic.
The anti-Salmonella monoclonal antibodies developed
according to the invention are truly monospecific and hence
can bs used without prior absorption. In contrast to the
2~7~2
conventional anti-Salmonella serotyping sera, the
monoclonal antibodies have much less batch~to-batch
variation in the process of production. Being truly
monospecific in kheir serological specificity, and having
less inherent variations from batch-to-batch preparation,
the monoclonal antibodies are therefore superior to the
conventional sera produced in experimental animals.
Another object of the invention is to provide an
improved laboratory diagnostic method for the detection of
salmonellosis. The advantage of the diagnostic method
according to the invention which uses enrichment serology
includes reduction of costs and time required as compared
to the conventional approach which relies on the isolation
and identification of Salmonella growing up as colonies on
agar media. The present method also has advantages over
other enrichment serology methods for the detection of
Salmonella in that:
(a) it is more specific than those described in the
literature since it uses a monoclonal antibody that is
directed against a genus-specific epitope present in the
outer core region of the Salmonella lipopolysaccharide
structure; and
(b) it has advantage over both the conventional culture
approach and the existing enrichment serology mPthod in
that it can also be adapted to carry out serogrouping of
the Salmonella at the same time as it is detected.
Therefore it can provide additional information as to the
type of Salmonella that is being detected and the
serogrouping step adds confirmation to the presence of
Salmonella in the sample.
A further object of the invention is to provide
comparative results obtained for the sample both before and
after suitable incubation in a medium that would allow the
target microorganism to multiply and hence the amount of
antigen to be detected to increase over time. As a result,
7 2~7~
the immunological method according to the invention permits
better discrimination of true positive findings from false
positive results which are common in immunoassays due to
non-specific stickiness of immunological reagents in the
test, especially when the test is done on biological or
food samples.
Accordingly, one aspect of the invention provides a
reagent for use in the serotyping of Salmonella, comprising
a panel of murine hybridoma anti-Salmonella monoclonal
antibodies against the Vi and the somatic O-antigens of
common serogroups of Salmonella which can be used without
prior absorption as factor specific sera, said panel
comprising one or more monoclonal antibodies selected from
the group consisting of M02, M04, C1-1, MO8, M09, E1-1, E2-
l and Vi; and said common serogroups including A, B, C1, C2,D1, E1, E4 and phage modified E1 Salmonella.
Another aspect of the invention provides a reagent for
specific detection of different common serotypes of
Salmonalla which comprises a murine hybridoma monoclonal
antibody directed against the genus-specific epitope of the
Salmonella lipopolysaccharide outer core region, said
monoclonal antibody consists of T6 murine hybridoma
monoclonal antibody.
A further aspect of the invention provides a method
for specific detection of different common serotypes of
Salmonella, which comprises, (a) treating a test sample in
an enrichment culture medium for incubation; and (b)
applying the resulting enriched test sample to a sandwich
capture ELISA assay with murine hybridoma monoclonal
antibodies directed against the genus-specific epitope of
the Salmonella lipopolysaccharide outer core region, said
monoclonal antibodies consisting of T6 murine hybridoma
monoclonal antibodies.
A further aspect of the invention provides a method
for simultaneously detecting and serogrouping Salmonella
8 ~7~
serotypes, which comprises applying sandwich capture ELISA
assay to an enriched test sample using unlabelled
monoclonal antibody T~ as capture antibody, and enzyme
labelled monoclonal antibodies that are directed against
the Vi or 0-antigens of serogroups A, B, C1, C2, D1, E1, E4
and phage modified E1 Salmonella as detector antibodies.
A further aspect of the invention provides a
quantitative immunoassay method for the detection of
Salmonella, which comprises: (a) treating a test sample in
an enrichment culture medium for incubation; (b) applying
the resulting enriched test sample to a sandwich capture
ELISA assay with a panel of murine hybridoma anti-
Salmonellalipopolysaccharidemonoclonal antibodies, either
against the Vi antigen or the somatic 0-antigens of common
serogroups of Salmonella and/or the genus-specific epitope
present in the Salmonella lipopolysaccharide outer core
region, as capture and detector antibodies, said panel
comprising one or more monoclonal antibodies selected from
the group consisting of Vi, M02, ~04, Cl~ 08, M09, El-l,
E2-l and T6; said common serogroups including A, B, C1, C2,
D~, E1, E4 and phage modified E1 Salmonella; and (c) taking
multiple time point ELISA measurements on the enrichment
culture at 0 hour of incubation and at subsequent time
: during the Salmonella culture step.
Yet a further aspect of the invention provides a
quantitative immunoassay method Por the ~etection of
infectious agents, which comprises taking multiple time
point ELISA measurements of a test sample undergoing
enrichment serology testing .in the presence of a specific
antiserum against the individual infectious microorganism
at 0 hour of incubation and at subsequent time during a
culture step.
2 ~
DETAILED DESCRIPTION OF T~E INVENTION
8eroloyiaal R~agents :
A panel of murine hybridoma cell lines that produce
specific and unique monoclonal antibodies against different
Salmonella antigens has been developed for improving the
laboratory diagnosis of salmonellosis. The specific anti-
Salmonella monoclonal antibodies include those directed
against the somatic O-antigens of the most common O
serogroups (namely those from groups A to E) as well as the
Vi antigen of Salmonella. Such a panel of anti-Salmonella
monoclonal antibodies is capable of use without prior
absorption as typing antisera for Salmonella.
The panel of murine hybridoma monoclonal antibodies
specific for different parts of the Salmonella somatic or
lipopolysaccharide (LPS) antigen has been developed for use
in immunoassays for Salmonella detection and/or typing.
These murine hybridoma monoclonal antibodies include: (i)
T6 which is directed against the common outer core of
Salmonella LPS; and (ii) those which are directed against
the Vi and the O-antigens of the most common O-serogroups
of A, B, C1, C2, D1, E1, E4 and phage modified E1 (Vi, MO2,
MO4, Cl-1, MO8, MO9, El-l, and E2-1). These hybridoma cell
lines have been deposited in the European collection of
Animal Cell Cultures, PHLS Centre for Applied Microbiology
& Research, Public Health Laboratory Service, Porton Down,
Salisbury, England (Cell Line Deposit ~ccession Numbers: T6
- 91070323, MO2 - 91070318, MO4 - 91071820, Cl-l
91070319, MO8 - 91070320, MO9 - 91070321, El-l - 91070322,
Vi6a - 91071816, and E2-1 - not yet deposited). These
murine monoclonal antibodies can be used without prior
absorption as either genus-specific or factor specific sera
for the detection and/or serotyping of Salmonella.
Mouse ascitic fluids induced by these murine hybridoma
cell lines can be used directly without absorption for
slide agglutination test to identify Salmonella.
.,
2~7~
The mouse ascitic fluid induced by the murine
hybridoma cell lines containing the monoclonal antibodies
should be titrated before use to find out the most optimal
amount or dilution for use in the slide agglutination test
using Salmonella organisms that are expected to give either
positive and negative slide agglutination reaction. The
diluted, ascitic fluid may be stabilized in diluent
containing preservative and may be stored at either -200 C
for long term storage or at 40 C. Positive and negative
control Salmonella microorganism should be used to test the
monoclonal antibodies at the beginning of each working day
or at least weekly for quality assurance purposes.
The slide agglutination test is carried out by
suspending a suspected Salmonella growing on a non-
selective agar medium in a drop (e.g. 10 ~1) of saline orphosphate buffered saline with or without phenol to give a
fairly dense milky suspension and 10 ~1 of suitably diluted
monoclonal antibody is added with mixing to the drop of
bacterial suspension. Then the slide is rock with gentle
rotation for 1 minute and any clear-cut clumpiny of the
bacteria is noted as positive agglutination. All positive
bacteria should also be tested with either saline or an
irrelevant monoclonal antibody in place of the specific
anti-Salmonella monoclonal antibody to check for auto-
agglutination of the Salmonella bacteria under test.
The monoclonal antibodies can also be used afterattachment to either protein A-Staphylococcal cells (which
are available commercially) or to inert latex particles
(also available commercially) after suitable purification
by DEAE ion exchange chromatography and/or Sepharcyl~ gel
filtration chromatography. In either case, this co-
agglutination approach would enhance the slide
agglutination reaction with positive Salmonella bacteria
and at the same time uses less amount of the specific
monoclonal antibodies.
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2~78~6~
11
An additional usage of these monoclonal antibodies is
to couple them to magnetic and/or immunomagnetic beads for
the specific separation and detection of Salmonella.
It should be noted that bacterial that exhibit
autoagglutination can not be tested by slide agglutination
method using either unconjugated ascitic fluid monoclonal
antibodies or monoclonal antibodies conjugated to either
protein A-Staphylococcal cells or latex particles.
Furthermore, as a general rule, serotyping of bacteria
should not be done on bacteria grown up on selective agar
medium because of possible false positive result.
Also, ascitic fluid store under harsh conditions (such
as exposed to excessive heat or been through repeated
freezing and thawing cycles) may loose their specific
reactivities with their corresponding Salmonella. When not
in use, the antibodies are best stored at refrigerator
temperature of about 4O C or for long term storage at -20O
C.
Nethod for Serotypinq and Detecti~g of Balmonella :
The use of murine hybridoma monoclonal antibodies
directed against the outer core region of the Salmonella
lipopolysaccharide as genus-specific polyvalent anti-
Salmonella sera for the detection of different common
serotypes of Salmonella is novel. Other serological
reagents that have bean employed for Salmonella detection
are either pools of polyclonal reagents directed against
the most common O-factors of Salmonella or are polyclonal
antiserum against poorly defined cell wall antigens (such
as the so-called "Common Structural Antigen, CSA") or
monoclonal antibodies against flagellar epitopes common to
different Salmonella. Howaver in the case of the latt~r,
the monoclonal myeloma proteins are not truly specific for
Salmonella since there are cross-reactions with another
member in the Family of Enterobacteriaceae, Citrobacter,
12 ~78t 62
and not all Salmonella are detected by such monoclonal
myeloma proteins.
For serotyping purposes, slide agglutination test with
diluted ascitic fluid induced by the hybridoma cell lines
or ascitic fluid antibodies attached to either protein A-
Staphylococcal cells or latex particles have been found to
be suitable.
The sandwich capture ~LISA can be done with unlabelled
monoclonal antibody T6 as capture antibody and enzyme
labelled monoclonal antibody T6 as detector antibody for
the detection of the most commonly encountered Salmonella
serotypes. If enzyme labelled monoclonal antibodies that
are directed against the O-antigens of serogroups A, B, C1,
C2, D1, E1l and E4 Salmonella are used as detector
antibodies, then simultaneous detection and serogrouping
can be done in the same test and the results obtained with
the enzyme labelled O-factor specific monoclonal antibodies
can also be used as a confirmation of the results obtained
with monoclonal antibody T6 alone.
The sampl~s to be tested (clinical material, food or
environmental samples) are put into a suitable enrichment
medium (this may include a pre-enrishment step in a non-
selective broth medium followed by incubation in a
selective enrichment broth medium for Salm_nella) for
incubation to allow even small numbers of Salmonella to
multiply to detsctable level by the immunoassay. Commonly
available enrichment media ~or the culture of Salmonella
include M-Broth (non-selective), Rappaport Vassiliadis
medium, Selenite-Cystine and Tetrathionate broths
(selective enrichment for Salmonella).
The sandwich capture ELISA is done using Dynatech
Immulon 2 microtiter plate coated with the ascitic fluid
monoclonal antibody T6. The optimal amount of T6 antibody
used for coating is determined by checker-board titration
and the diluent for diluting the T6 antibody for coating is
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~7~ 2
13
carbonate-bicarbonate buffer, pH 9.6 (1.59 g Na2C03, 2.93
g NaHC03, in one litre of distilled water). Wells are
filled with 100 1 of the T6 antibody for coating at 4O C
overnight. Following three washes with phosphate buffered
saline (PBS) with 0.05% Tween 20 (PBS-Tween), wells are
blocked with 300 1 of 2% bovine serum albumin (BSA) in PBS
at 37O C for 1.5 hour. After three more washes as above,
samples are added at a volume of 100 1 per well for
incubation at 37~ C for 1 hour. Then wells are washed four
more times as above and Salmonella antigens captured are
detected by suitable dilution (determined by titration) o~
enzyme (e.g. horseradish peroxidase) conjugated monoclonal
antibody T6 using 100 1 per well for incubation at 37O C
for 1 hour. After a final wash of four times as above,
wells are developed by addition of 100 1 per well of 0.1
mg/ml of O-phenylenediamine dihydrochloride (OPD) in a
substrate buffer made up of 0.05 mol/l Na2HP04, 0.025 mol/l
citric acid with 0.003% H202. The colour development is
carried out in the dark at 37O C for 30 minutes, and the
substrate-enzyme reaction is stopped by addition of 25
per well of 4N H2S04. The colour developed is read with a
microtiter plate ELISA reader (e.g. Dynatech MR 710
microtiter plate reader) at the wavelength 492 nm using
suitable filter. Controls including standard amounts of a
Salmonella Ra-LPS as well as overnight broth culture~ of
any common Salmon~lla serovars are included together with
buffer without any antigen or Salmonella serving as
negative controls. In deciding on an optical density value
~or calling a test sample as positive, mean optical density
value from no antigen control experiments plus three times
their standard deviation is used. This will allow for more
than a 95% confidence limit.
Under normal testing condition, it is recommended that
samples be tested in triplicates. Therefore in a 96 wells
microtiter plate, 32 samples of unknown, positive and
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;
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2~7~162
14
negative controls can be accommodated in one run on a
single plate.
For simultaneous detection and serogrouping of
Salmonella, samples are set up in multiple wells so that
some wells will receive enzyme labelled monoclonal antibody
T6 while other wells will receive enzyme labelled anti-
Salmonella 0-antigen specific monoclonal antibodies.
However, it is worth noting that genuine cross-
reactions of the monoclonal antibodies developed against
Salmonella lipopolysaccharide antigens with non-Salmonella
bacteria may create false positive results. For example it
is known that monoclonal antibody T6 will react with
bacteria having the disaccharide N-acetyl-D-glucosamine
alpha 1,2 linked to D-glucose as in the Escherichia coli
having the R2 core type. However such Escherichia coli has
been found in only a couple of serotypes that are not
commonly encountered.
Also, failure to grow up sufficient numbers of the
Salmonella organisms in a suitable enrichment broth media
may lead to false negative results.
Multi~le Time Point Immunoa~Yay:
The immunoassay approach that follows the kinetics of
the growth of the target infectious microorganism by taking
multiple measurements during the culture step of the target
microorganism is new and add~ advantage to the existing
method by including a self control for each specimen as a
reference point to compare results with. This feature
serves as a control for non-specific reactions that are
prone to occur in immunoassays done on biological as well
as food and environmental samples.
By performing the immunoassay or the target
microorganism both at 0 hour of incubation and at different
times during culturing of the sample for the target
organism in a suitable broth medium allows for accurate
2~7~62
documentation of the presence of viable target
microorganism. After the base line 0 hour measurement has
been noted, the subsequent frequency of taking a sample out
from the broth culture to do immunoassay will depend on the
specimen type and the anticipated number of target
microorganism that may be present in the sample as well as
the detection limit or sensitivity that one aims to
achieve. In practice, at least two samples should be
taken, one at the 0 hour of incubation and one at tha end
(preferably after overnight incubation of the broth medium
to allow detection of even very low numbers of the target
microorganism) of the incubation period for culturing of
the target microorganism. If more than two assays can be
done on a single sample, then they should include at least
the two time points as described above plus samples that
have been incubated 4 to 6 hours allowing adequate time for
target microorganism multiplication.
This novel approach offers an easy method to
discriminate any false positive results due to non-specific
stickiness of the immunological reagents in the test from
the genuine positive results due to the presence of
Salmonella as the latter would increase during incubation
of the culture while the former would only remain static.
This approach can also be extended to the deteation of
other infectious ayents with enrichment serology method,
provided that specific antiserum against individual
microorganism is available. Other immunoassay methods such
as competitive assay can also be used with this approach.
This applies to the nature of the anti-serum too.
Polyclonal sera instead of monoclonal antibodies can also
be used with this method.
However, it has been found that non-physiological pH
and ionic condition of the assay medium may lead to
spurious reactions.
~7~2
Embodiments of the invention will now be described, by
way of laboratory or commercial tests results, with
reference to the accompanying drawings, in which:
Figure 1 shows standard dose-response curves for the
detection of Salmonella typhimurium Ra and smooth LPS.
~A) S0rotyping of Salmonella with Nurine Nonoclonal
Antibodies:
Table 1 describes the eight murine anti-Salmonella
hybridoma monoclonal antibodies, their isotypes and
agglutinating titers against homologous serogroup of
Salmonella. The slide agglutination reaction is in ~ll
cases very strong and occur within on~ minute of mixing the
diluted ascitic fluid with the bacterial suspension.
The specificity of the monoclonal antibodies is
demonstrated by testing the antibodies with standard
strains obtained from thP American Type Culture Collection
(ATCC), Rockville, Maryland, U.S.A. as well as clinical
isolates of Salmonella obtained from a university teaching
hospital in Hong Kong.
17 ~7~2
.i.b.le 1. Isotypes and agglut;nating titers of mouse ascitic
fluid monoclonal antibodies against somatic 0-antigens
~,f serogroups ~ to E ~alrnclnella
....______________________________________________________________
Monoclonal SerogroupSlide ac~,~c,lutinationntibod~ specificity Isotype titer agair,st homologGus
seroc,roup of Salmonella
__ ________________ ____ _____________________ _____ _______~
M0? ~ IgGl1:32
~Q4-4 S Ic,Gl1:16
Cl-l ~L IgG31:'2
~10S C2 IgG31:32
MG~ Dl I gGl1: :~52
El-l ElfE4 IqGl. 1:64
*
E -l E2,fr-3 IgGl1:3~
:
Vi Vi antigen IgG2b1:32
of Salmonella
and Citrobacter
__ ____________ __._______ __ __ ___ ___ ________________ ____
*
strains of serogroups E2 and E3 are phaga modified serogroup
El bacteria, an~l they are nol~i classifieci as serogroup E
strains. E2 and E3 are previous designations.
. .
2~7~62
18
Amongst the 13 non-Salmonella coliform ATCC standard
strains, none was found to be agglutinated by the anti-
Salmonella monoclonal antibodies. In contrast, 5 standard
Salmonella strains obtained from ATCC were all agglutinated
by the anti-Salmonella monoclonal antibodies according to
their serogroup specificity (Table 2).
19 2~7~2
Table 2. Spe'ci~icity c,f the monoclonal antibodies againsL somatic
0-antigens o~ serogroups ~ to E ,S,,.,~,.l,"m""o,n,e.,,l,,l,"a", teste~ by slide
a~91 U Li nation with ~TCC~ ~strains
______________________________________________________________________ ,
Salmonella
Sero- ' ~gglutination with
group Serotype ~TCC No M02 M04 C~ 08 M09 E1-1 E~-1
___ _________________________________________________________________
S paratyphi ~ 9150 ~ - - - - - - ;
C1 S choleraesuis 13312 - - +
C2 S. newport ~962 ~- - - +
D1 S. enteritidis 13076 + - - - . ~ - -
S. typhi 19430 - - - - ~ - -
.~ . .. . . .. = . ...
Non-Salmonella ~TCC No~ , . -
specles
Escherichia coli 25922 - - - - ~ - - -
Escherich;a coli 35~18
Shigella fle~neri 29903 '
Shigella sonn~i 25931
Klebsiella pneumoniae 13883
Enterobacter 27155 - - - -
a~lomerans
Enterobacter,cloacae 23355 - - - .- , .-
Proteus mirabilis 7002 - - - . .-
Providencia.rettgeri 29944 - - - '-'
~ Citrobacter freundii 8090
Serratia marcescens 8100
Edwardsiella tarda 15947
Yersinia enterocolitica 9610
____ _ _ ___ __ ______________________________ ________ ___________
Strains were purchased from Rmerican Type Culture Collection..
M02 and M04 were used at a dilution of 1:8~ while C1-1, M08~ M09,
E1-1 and E2-1 were used at a dilution of 1:16
- . ,
2~J7~1~2
Further studies with clinical wild isolates from
patients suffering from salmonellosis substantiated their
specificity (Table 3).
2~7~
21
Table ~ Sero~yping of 10~ clinlcal isolates of ,',~,c~ln!on~,l,l,,a, by slid~-
agglutination .est uslng a panel or anti--,S"al"monel,,l,,a mono
clonal antibodies
______________________________ _____________________________________
:K `t
~ero- Serotype No of ~gglutinatlon with r~bs :
group stl-alns MO M04 Cl-l riO~ MO~ El-l E2-1.
tested
..____________________________________________________________________
S paratyphi ~ S ~- ~
- C S typhimurium 17 - +
S d~rby 13 - +
S~ sa;ntpaul 4 - +
S agona ~ - +
' ~. paratyplli' S 1 . - +
01 S infantis 8 - -
S. braenderup, ,, 1, , - , -
S thompson 1- - -
C2 S manhattan 14 - -
'' S litchfield 4 , - -
S chailey ~ - -
S bloc~ley 2 - -
S newport 2 - -
Dl S. enteritidis 6 + -
S panama 2 ~
S~ durban , 1 - -
S typhi~ 2" +~- -
.. ,: . .. .. . . . .. .
Ei S, anatum S - - - - - +
'S~ weltevreden 3 - - - - - +
S meleagridis 1 - - - ~ ~ + ~
E4 S krefeld ~ - - - - - +
E2 S portsmouth 4 - -' - - - -
____________________________________________________________________
Total No = 107
*
Serogroup and serotypes of S"al,"moc,el,l,a, were identified by slide
agglutination test using commerical ,S,,,,a,l,m"o,n,e,ll,,,a agglutlnatln~
, antisera.
+
M~bs M02 and M04 were used at a dilution of 1:8, and M~bs Cl-l~ ~08,
MO97 El-l and E2-1 were used at a dllution of'l:l6 in the slide
agglutination test.
One strain of S~, typhi was agglutinated by both M02 and M09 while
the other strain of S.,_,t,y~h,i was agglutinated by ~09 only
.... ,.. - - - :
22 2~7~2
The only result that did not show concordance with the
conventional polyclonal antisera was found with the
mono~lonal antibody M02 which is specific or the
dideoxyhexose paratose linked to the backbone somatic 0-
antigen of serogroup A almonella. In the study of theSalmonella clinical isolates, it was noted that monoclonal
antibody M02 which besides ayglutinating the serogroup A
Salmonella was also found to agglutinate some serogroup D
strains. In closer examination of the serogroup D1
Salmonella, it was found that these strains show close
resemblance to those belonging to serogroup A. The major
difference lies in their specific 0-antigen which is
specified by the dideoxyhexose, paratose in serogroup A and
tyvelose in serogroup D. Furthermore the biosynthetic
pathway of these two dideoxyhexoses is also inter-related
in that the two sugars can be interconverted by an
isomerasa enzyme which is present in the serogroup D
Salmonella. Hence it is not surprising that stains of
serogroup D Salmonella may express the dideoxyhexose
paratose in its cell surface which may then be detected by
the monoclonal antibody M02. Nevertheless this unusual
cross-reactions between the serogroup D Salmonella with the
M02 monoclonal antibody does not seem to cause confusion in
the differentiation of these two serogroups of organisms
since the cross-reactions seems to occur in one direction
only and there is no similar cross-reactions of serogroup
A Salmonella strains with the M09 monoclonal antibody.
Therefore strains of Salmonella reacting with M02 alone
belongs to serogroup A while strains reacting to either M09
alone or both M09 and M02 belong to serogroup D.
Further evaluation of the anti-Salmonella monoclonal
antibodies as serotyping reagents for Salmonella have been
carried out at the German National Salmonella Centre at
Hamburg, Germany and at the National Laboratory for Enteric
Pathogens of the Laboratory Centre for Disease Control,
~7~.62
23
Health and Welfare Canada, Ottawa, Canada. Part of the
evaluation done in Germany is summarized in Table 4.
TABLE 4 Serotyping of Groups Cl and C2 and Other Related O Serogroups
of Salmonella by Monoclonal Antibodies Cl-lICI-2 (Specific for
Group C~) and M08 (Specific for Group C2) in the National
Reference Center for Salmonella, Ger~nany
Agglutinating Results with Different Dilutions o~
MAbs'
Cl-1 C1-2 M08
Test Strains 1:101:20 1:301:10 1:201.301:10
S. choleraesuis
6~,7:-:1,5 + + + + + + + + + + + +
62,7:-:1,5 + + + + + + + + + (+
S. Iomita
62,7:e,h:1,5 + + + + + + + + + (+)
S. thompson
6l,2,7:k:1,5 + + + + + + + + + +
S. mantevideo
6t,2,7:g,m,s:- + + + + + + + + + + +
S. oranienburg
6~,2,7:m,t:- + +. + + + + + + + + + .
S. bareilly : .
6l.2,7:y:1,5 + +. + + + + + + + + + +
S. infantis
6,12,7:r:1,5 '+ + + + + + + + + +
S. virchow
6~,2,7:r:1,2 ' + +,+ + `'+ + + + + + +
S. oslo
6, 2,7:a:e,n,x + + + + + + + + + + + +
S. paratyphi C
6l,2,7,Vi:c:1,5 + + + + + ~ + + + + +
S. georgia
6J,2,7:b:e,n,z~s + + + + + + + + + +
S. potsdam
6~ 2,7:1,v:26 + + + +
S. braenderup
6~,2,7:e,h:e,n,z,5 + + + + + + + + + + +
5. II 6~.2,7:b:z39 + + + + ++ + + + (+
S. II 6~,2,7:g,m,s,t:- + + + + + ++ .~ + + +
.
Gtoup C, 0:14 +
S. ohio
6,7,14:b:1,w + .~ + _ + + (+
S. eimsbuttel
6,7,14:d:1,w + + + + _ + + + _ _
24 2~7~62
TA~LE 4 C~ntinued
- i
Agglutinating Results with Different Dilutions of
MAbs~
~ 1 C1-2 MO8
Test Strains 1:10 1:201:30 1:10 1:20 1:30 1:10
S. livingstone
6,7,14:d:1,w ++ + _ ++ +
S. nieukerk
6,7,14:d:7~ + + + _ + + (+
S. rnontevideo
6,7,14:g,m,s:- + + + - + + +
S. oranienburg
6,7,14:m,t:- + + + _ + + +
S. thompson .
6,7,14:k:1,5 + + + - + + +
S. infantis
6,7,14:r:1,5 + + + - + + +
S. Iille .
6,7,14:z38:- + + + ; ~ + + (~) ~ ~
5. II " ,
6,7,14:g,m,s,t:- + + + + ....... - + + +
Arizona 0:6,7
5. IIlb 6,7:1,v:z53 + + + _ + + +
5. IV
6,7:z~,z23:- + + + (+) + + +
S. Vl
6,7: ~:1,7 + + + (+) + + +
Group 0:18
S. rawash
6,14,18:c:e,n,x + + + (+) + + (+)
S. usumburu
6,14,18:d:1,7 + + + (+) + + (+)
S. cerro
6,I4,18:z~,Z23:- + + + (+) + + (+)
. .;
-- Cl-1 a-2 MO8
1:10 1:10 1:10 1:20 1:30
Group O:H
5. carrau
6,14,24:y:1,7 - - Not done
S. bahrenkld
6,14,24:e,h,:1,5 - - Not done
S. albuguerque
1,6,14,24:d:z6 - - Not done
S. onderstepoort
1,6,14,24:e,h:1,5 - - Not done
S. boecker
1,6,14,25:1,v:1,7 -- -- Not done
S. fischerkietz
1,6,14,25:y:e,n,x - - Not done
S. uzaramo
1,6,14,25:7l,z2~:- -- -- Not done
S. lIlb
6,14:b:e,n,x ~ ~ Not done
TABLE 4 Continued . 2 7 16
- -- V o
Agglutinating Results with ~ifferent Dilutions of MAbs~
Cl-1 C1-2 M08
1-10 1:101:101:20 1:30
S. IV
6,14:z~,zu:- - - Not done
5. Il
1,6,14:Zlo:1,5 ~ -- - Not done
Group 0:8 (=C2)
S. muenchen
6,8:d:1,2 - -- + + + + (+
5. manhathn
6,8:d:1,5 _ _ + + +
5. neu~ort
6,8:e,h:1,2 _ _ + + +
6,8:m,t:1,5 - - + + +
5. blockley
6,8:k:1,5 -- -- + + +
5. Iitckfield
6,8:1,v:1,2 . -- -- + + +
5. rnanchester
6,al,vl,7 , - - ++ +
5. bovtsmor~ificans . + + (+)
5. goldcoast
6,8:r:1,w _ .
6,8 Z~C:e,n,x -- -- + +
5. hiduddih
6,8:1,z~,z~3:1,5 -- -- + + + (+)
5. kottbus
6,8:e,h:1,5 - - + + +
6,8 z~,Zu:e,n,Zl~ - -- + + + ( ) '~
5. herston
6,8:d:e,n,z~5 - - + + + (+)
6,8:zz9:e,njx - - + + + ( + j
8 k 1 5 - - - Not done
S. kentucky Not done -
S. amherstiana _ _ Not done
8,20.g,m,s:- -- -- Not done
5. enteritidts Not done
5. senftenberg Not done
5. aberde~t Not done
3,10-.1,v:1,6 -- -- Not done
1,13,22:z:1,6 - - Not done
.:
26 2~7~162
TABLE 4 Continued
I
Agglutinating Results with Different Dilutions of MAbs'
Cl-1 Cl-2 MO8
1:101:10 1:10 1:20 1:3Q
5. waycross
41~ Not done
S. uccle
54:g,s,t:---- -- -- Not done
~ , quick and strong positive reaction; +, positive agglu~nation; (+)~ late positive agglutin-
ation: and -, negative agglutination.
. . .
~;
-- . .
27 2~78:~fi2
(B) Detection of different common serotype~ of Salmonell~
by ~andwich capture ELIS~ technique using murine
monoclonal antibody directed against the genu8-
speaific apitope of the Salmonella lipopolysaacharide
outer core structure:
The sandwich capture ELISA technique using monoclonal
antibody T6, which is directed against the genus specific
epitope found in the outer core region of the Salmonella
lipopolysaccharide molecule, has been found to be able to
detect both smooth and rough lipopolysaccharide antigens of
Salmonella and that the method is reproducible as shown in
Figure 1. The antibody (T6) and the method ~sandwich
capture ELISA) using monoclonal antibody T6 are both
specific for the detection of Salmonella organism since 21
non-Salmonella standard strains obtained from American Type
Culture Collection tested did not give positive result even
when tested at a number of >109 per ml (e.g. overnight
broth culture in Brain Heart Infusion medium).
In contrast to the overnight broth cultures of non-
Salmonella bacteria which uniformly gave negative result in
the sandwich capture ELISA test with monoclonal antibody
T6, four hour broth cultures of seven standard Salmonella
strains and 24 clinically isolated wild Salmonella strains
from the common O serogroups of A to E were all detected by
this method (Table 5).
2~17~ 62
28
. ~ ~ ,~ _
`'C--1 . _ + 't + + + ~ + t- + + + + + + T + + + + + + + T +
tr)~1:~ _ _ ~
e ~- ~ C~ ~ ~ r' ~ r- ~ o g ~o o -r o = ~ r~
u :~ o ~ _ O O O _ O O O ~ O O O O c~ O O O _ O _ _ _ O O
c . .
a a ~ , 3 ~ 3 ~ ~ ~ a ~ ~ ~ 3 E ~ a ~ E
6 ~
c O
u cc
~ 0~ tq ~ ~ a ~
c _ _
. ~. ~ + + ^ + + + + I î I î I I I I I I I t
r ~ O ' o ~ O ~ ~ ~ ~ ~ O O ~ O O o O ~ ~ o ~
o~ ~ C~ O ~ ~ O g 00 0 ~ U~ ~ ~ ~ ~ _
v~ o ~ o Ch U~ ~_
e ~ ~ ~ k ~ ~ ..
¦ ~
~ ~ 7 ~
29
Using a combination of anti-Salmonella o and LPS core
specific monoclonal antibodies in the sandwich capture
ELISA, it has been demonstrated that not only can
Salmonella strains be detected, their serogroup identity
can also be found out at the same time (simultaneous
serogrouping). Table 6 illustrates the results obtained
using horseradish peroxidase enzyme conjugated monoclonal
antibodies M02, Cl-1, M08, MO9 and E1-1 as detector
antibodies and monoclonal antibody T6 as capture antibody
in the sandwich capture ELISA test for the detection of
Salmonella.
~7~2
Table 6. Simultaneous detection and serogrouping of Salmonella using
a combination of anti-O and anti-core specific monoclonal
antibodies in sandwich capture ELISA.
______ ______________________________ _______ _______________________
Mean ELISA O.D. and (Reactivity) for:
Salmonella strains HRPO conjugated monoclonal antibodies
(serogroup) MO2 C1-1 MO8 MO9 El-l
_________.__________________________________________________________ _
S. paratyphi A 0.~70 0.005 0.004 0.056 0.006
(serogroup A) (+) (-) (~) (~) (~)
S. typhimurium 0.016 0.028 0.029 0.014 0.025
(serogroup B) (-) (-) (-) (-) (-)
S. choleraesuis 0.041 0.995 0.038 0.013 0.034
(serogroup Cl) (-) (+) (~) (~) (~)
S. newport 0.024 0.029 1.011 0.014 0.021
(serogroup C2 ) (-) (-) (+) (-) (-)
S. enteritidis 0.038 0.009 0.016 1.057 0.009
(serogroup Dl) (-) (-) (~) (+) (~)
S. anatum 0.005 0.012 - 0.013 0.013 0.933
(serogroup El) . (-) (-) (-) (-) (+)
_____ _ _ _ ______________________ __ _________ __________________ _
Escherichia coli 0.035 0.057 0.049 0.006 0.034
Brain Heart Infusion 0.004 0.000 0.010 0.019 0.012
medium
___________________________________________________ ________________
*
mean of triplicate determinations
31 2~7~
~C~ Deteation of almo~ella in different specimen3 using
multiplo time point immunoassay:
~i) Detection of Salmonella enteritidis phage type PT 8 in
egg specimens:
Homogenized grade A eggs were first tested for the
absence of Salmonella using standard bacteriological
culture techniques. After assuring that no Salmonella was
present in the egg specimens, different numbers of S.
entexitidis PT8 was used to spike the homogenized egg.
ml of spiked egg specimen was inoculated onto 9 ml of
Selenite Cystine broth for incubation at 37~ C. Samples
were removed at different time periods (0, 4, and 18
hours), boiled for 30 minutes and assayed in the sandwich
capture ELISA.
The results of Table 7 indicates that the sandwich
capture ELISA using monoclonal antibody T6 can detect less
than 10 S. enteritidis present in egg specimen after
suitable enrichment culture in Selenite Cystine broth
medium. Similar results were obtained with another isolate
of S. enteritidis PT4 (data not shown).
' 32 2~7~ fi2
Table 7. ~etection or S, ,en,teritidis phage type ~ in eggs
using sandwich cap~ure ELIS~ with murine monoclonal
antibod~" T6 using a multiple time point assa~
Inoculumn size ~ean ELIS~ O.D. obtained at: 1
used to spike 0 hour 4 hour 1~ hour
eggs f~c~u)*
~._______ ____ ______________________________________________
Norle 0~09~ 0~076 0 100
1.25 x lO6 0.104 1 47~ 1.570
1.25 x 105 0.0~1 0~322 1 ~0
1.25 ~ 104 0 0~ 0.072 l.45.`s
I.25 x 103 , 0 039 0 059 1 553
L.25 x 102 O.lOl 0.065 1.462
1 25 x 10' 0~0~3 0 061 1.447
. 25 0.0~7 0.074 1.26~
0 125 O lY~2 0.0~9 0 10~ ',
I
______,_ ___ ____ __________________ _
- * col ony formi ng uni ts
..
~7~1 ~2
(ii) Detection of different serotypes of Salmonella in
faecal specimens obtained from patients suffering from
gastroenteritis:
specimens were tested immediately (O hour
incubation) by sandwich capture ELISA after inoculation
onto Selenite Cystine Broths as well as after overnight
incubation at 370 (Table 8).
Table 8 ~etection of S,a,l,m,one,l"l..a, from human faecal specimens
by sand~ich capture ELIS~ ~Jith monoclonal antibody
T6 using a multiple time point assa~f
Specimen ~ean ELIS~ O D. reading after Culture for
~umber 0 hour overnight incubation Sal.rnonella
_______________________________________ __ _______~_____ , .
~43C,~ o oC~o O.f99 positive
64839 0 0670~387 positi~fe
3 0 2521~15~ positivP
61474 0 6100 911 negative
~'~2914 b 223 0.176 - ' negative
. .. ..
- ~3620 0 9~0 945 negativ~
~3871 '0 5360 575 negative
61057 0 0390 044 negativci
~1664 0.034 0.052 negative
64619 0.0420~055 negative
.
__ ___________________________________________________________
- . .... .. . .
2~7~ ~2
34
The results of Table 8 clearly shown that for some
specimens, if only a single assay was done, say after
overnight incubation, the high reading obtained would have
suggested the presence of Salmonella. However high reading
was already found in the specimen without any incubation,
and standard bacteriological culture done on the specimen
both before and after the incubation in Selenite Cystine
Broth had not provided any positive isolation of
Salmonella. Therefore the high ELISA reading obtained at
the O hour of incubation of the enrichment broth as well as
after overnight incubation most likely was due to some non-
specific reaction unrelated to the presence of Salmonella.
Specimens with positive isolation of Salmonella all gave
significantly increased (at least two folds) ELISA O.D.
between the two time-point measurements.
