Antibodies to S. mutans and uses thereof

Details Antibodies to S. mutans and uses thereof Antibodies to S. mutans and uses thereof

1999-08-20

2001-05-15

Graser, Jennifer (Department: 1645)

Drug, bio-affecting and body treating compositions

Immunoglobulin, antiserum, antibody, or antibody fragment,...

Binds bacterium or component thereof or substance produced...

C424S130100, C424S141100, C530S388100, C530S388400, C530S388200, C435S243000, C435S325000, C435S326000, C435S332000, C435S354000

Reexamination Certificate

active

06231857

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to novel antibodies to the
Streptococcus mutans
bacteria that are naturally found in the mouth, and play a role in the development of dental caries. The invention relates to methods of detection of
S. mutans
using the antibodies of the invention or fragments or derivatives thereof. The invention also relates to diagnosing, monitoring, treating and protecting the teeth from dental caries using the antibodies of the invention or fragments or derivatives thereof.
Throughout this application, various publications are referenced within parentheses and cited at the end of the application. The disclosures of these publications are hereby incorporated by reference herein in their entireties.
Currently human dental caries, or cavities, are detected by changes in translucency, color, hardness or X-ray density of teeth. These technologies have limitations both in specificity and reproducibility. Further, they do not show, at a single time point, whether or not the disease is active.
The bacterium
Streptococcus mutans, or S. mutans
(named and described by Clark in 1924) is known to be a prime etiologic agent for the initiation and progression of human dental caries. (Fitzgerald and Keyes, 1960; Loesche, 1982; Loesche, 1986; Tanzer, 1997).
S. mutans
is one of the primary factors in acid dissolution of the apatite (mineral) component of the enamel then the dentin, or of the cementum then the dentin. A strong correlation between the proportion of
S. mutans
in dental plaque or in saliva relative to other bacterial species and the presence or risk of future outbreaks of dental caries has been documented, (Tanzer, 1997; Krasse, 1988). Therefore,
S. mutans
in plaque or saliva can serve as an index for both caries activity state and caries risk or susceptibility (Loesche et al., 1975; Ellen, 1976; Krasse, 1985; Krasse, 1988). These indices play an increasingly important role in the diagnosis and treatment of dental caries (Hume, 1993; Mundorff et al., 1993; Van Houte, 1993).
Present techniques of detection and quantitative determination of
S. mutans
include bacterial culture with selective media using either broth or agar plate systems (Ellen, 1976; Loesche, 1982) and polymerase chain reaction techniques (Igarashi et al., 1996). Each of these methods requires significant time (on the order of days), well trained personnel and sophisticated equipment to perform. Consequently, existing techniques are relatively expensive and time consuming.
Alternatively, monoclonal antibody based detection methods allow a rapid and accurate, yet economic quantitative measurement of the presence of bacterial cells, and have significant advantages compared to traditional culture sensitivity assays or polymerase chain reaction (PCR) techniques. Bacteria produce unique polysaccharide structures of lipooligosaccharide or lipopolysaccharide or other polysaccharides, among a large range of other chemical components and products, on their cell surfaces. Monoclonal antibodies can be raised against these chemical structures, using standard hybridoma techniques (Kohler and Milstein, 1975). The sensitivity and accuracy of this method is largely dependent on the specificity of the monoclonal antibody produced. By making and screening large numbers of hybrid cell lines, one can find certain monoclonal antibodies which are species-specific for the desired bacteria, i.e. the monoclonal antibodies ICL
11
and ICL
12
recognize the 0139 antigen of
Vibrio cholerae
(Hasan, 1994)). These monoclonal antibodies can be linked to various detection systems including, for example, fluorescent reagents, calorimetric reagents or coagglutination reagents. The resulting labeled antibodies can specifically bind to the desired bacterium in any sample, and rapidly present the result through the linked detection systems (Harlow and Lane, 1988).
TABLE 1
monoclonal
PCR
Culture Sensitivity
antibody
Reporting
2 days
7-10 days
3-10 min
time
send out samples
send out samples
instant results at
chairside
Cost
~$100
~$100
less than $10
Precision
Species level
genus level
Species level
Test
few hundred cells
only cultivable cells
few hundred cells
sensitivity
regardless of
need viable cells
regardless of
viability
viability
Accuracy
>90%
~50%
>90%
Lab
yes
yes
no
requirement
Due to overwhelming advantages, monoclonal antibody-based detection methods have been widely used in medical microbiology. At present, there are nearly one hundred different monoclonal antibody based detection methods available to diagnose various pathogenic bacteria. However, to date there have been no such methods available for the detection of dental caries. While other investigators have made monoclonal antibodies to
S. mutans
, these monoclonal antibodies are not suited for diagnostic or clinical uses because they are not species specific for
S. mutans
and cannot detect the presence of
S. mutans
at very low levels. Almost all previous monoclonal antibodies were made against surface proteins of
S. mutans
(i.e. glucosyltransferase, agglutinin, surface antigen PI, etc.). Similar proteins can also be found on other Streptoccocci species. Thus, these monoclonal antibodies can not be used to distinguish
S. mutans
from other Streptoccocci species. To diagnose dental caries it is important to be able to discern the
S. mutans
strain from other bacteria that may be present. One needs to find a monoclonal antibody that is specific for a unique epitope specific to the
S. mutans
cell surface.
SUMMARY OF THE INVENTION
Accordingly, the invention describes three monoclonal IgG antibodies, referred to as SWLA
1
, SWLA
2
, and SWLA
3
, which appear to recognize a species-specific polysaccharide on the cell surface of
S. mutans
. The invention also describes a rapid method of detection of
S. mutans
without the need for prior growth of bacteria in culture. The invention further describes methods of utilizing these antibodies for rapidly quantitatively detecting
S. mutans
. These methods are sensitive enough to detect the presence of a single
S. mutans
bacteria cell. These methods can be widely used in the clinical diagnosis and treatment of dental caries. Yet another embodiment of the invention is a diagnostic kit containing the monoclonal antibodies of the invention and reagents for detecting binding of the antibodies to
S. mutans
cells on teeth in or in a sample from a subject.
In particular, one aspect of the present invention is a monoclonal antibody that specifically binds an antigen on the surface of
Streptococcus mutans
and which is the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB12559, and which is designated SWLA
1
.
Another aspect of the present invention is a monoclonal antibody that specifically binds an antigen on the surface of
S. mutans
and which is the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12560, and which is designated SWLA
2
.
Yet another aspect of the present invention is a monoclonal antibody that specifically binds an antigen on the surface of
S. mutans
and which is the monoclonal antibody produced by a hybridoma deposited with the American Type Culture Collection as ATCC No. HB 12258, and which is designated SWLA
3
.


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