WO1997005486A1

WO1997005486A1 - Rapid immunoassay for streptococcus mutans

Info

Publication number: WO1997005486A1
Application number: PCT/US1996/012135
Authority: WO
Inventors: Stephen Alden Ralls; Llyod Grant Simonson
Original assignee: The United States Of America, Represented By The Secretary Of The U.S. Department Of The Navy
Priority date: 1995-07-28
Filing date: 1996-07-23
Publication date: 1997-02-13
Also published as: EP0871890A4; EP0871890A1; AU6507896A

Abstract

An assay for detecting oral bacterium, preferably Streptococcus mutans, most preferably serovars c, e, and f, comprising gathering a sample suspected of containing an oral bacterium of interest (target), immobilizing any oral bacterium present on a non-inactive substrate, contacting the sample with an antibody, either polyclonal antibodies (absorbed animal antisera) or monoclonal antibodies, that are specific for the sought or target oral bacterium; contacting the antibody with a label capable of being detected thereby identifying the presence of the antibody, detecting the label, whereby the presence of Streptococcus mutans in the media is determined. The device for conducting these tests is a frame or support holding non-interactive material capable of binding the sought antigens of interest (target) while permitting drainage of fluids.

Description

RAPID IMMUNOASSAY FOR STREPTOCOCUSS MUTANS

Field ofthe Invention

This invention relates to a rapid assay method and means of detecting dental caries.

More particularly, the invention relates to a rapid assay test for detecting serovars of

Streptococcus mutans.

Background ofthe Invention

Certain species of oral bacteria have been associated with dental caries in humans (1).

The occurrence of Streptococcus mutans is highly correlated with dental caries and this has

been widely addressed in the dental literature (1-17). Serotypes (serovars) c, e, and f of

Streptococcus mutans have been described (17). Ebersole has described a SEROLOGICAL

METHOD FOR THE IDENTIFICATION OF MICROORGANISMS in U.S. Patent N°

4,458,014 specifically for the identification of diseases of the mouth. Chen et al. describes

a DETECTION OF HUMAN ORAL CELLS BY NUCLEIC ACID HYBRIDIZATION to

detect the same target organisims as are targeted in this invention. Both the methods of

Ebersole and Chen are time consuming and are not rapid. The use of antisera to Streptococcus

mutans as a chairside risk assessment product (rapid diagnosis) has not been reported. The

detection of these serovars will help to identify the presence of caries long before the pitting

and other physical indicators of caries are evident and at a rate faster than now available

through Chen et al. , Ebersole or Rosenberg et al. U.S. Patent N^a 4,976,951, DENTAL

CARIES DIAGNOSTIC AND LOCALIZATION TECHNIQUE.

Previous methods can detect Streptococcus mutans levels in 48 hours using dental

plaque (U.S. Patent #3,746,624) or saliva (Dentocult SM Strip Mutans, Vivacare diagnostic

line/ VTV⁷ ADENT, manufactured by Orion Diagnostica, Espoo, Finland). These older methods are considered too slow, particularly by persons who are traveling to distant locations were

dental services are difficult to obtain and by dentists seeking immediate answers for patients

in their care. What is needed is a simple to operate, rapid test that can be developed and read

in less than an hour. SUMMARY OF THE INVENTION

Accordingly, an object of this invention is an immunodiagnostic assay for rapidly

detecting the presence of Streptococcus mutans (especially serovars c, e, and f) in human

dental plaque and/or radiographic Lesions.

Another object of this invention is a diagnostic method for rapidly assessing dental

caries risk.

A further object of this invention is a diagnostic method for rapidly determining whether a radiographic carious lesion is in an active disease state (positive for a certain level

of Streptococcus mutans).

An additional object of this invention is a device for conducting the rapid immuno

assay.

These and additional objects of the invention are accomplished by an assay for

detecting oral bacterium, prefereably Streptococcus mutans, most prefereably serovars c, e,

and f , comprising gathering a sample suspected of containing an oral bacterium of interest

(target), immobilizing any oral bacterium present on a non-inactive substrate, contacting the sample with an antibody, either polyclonal antibodies (absorbed animal antisera) or monoclonal

antibodies, that are specific for the sought or target oral bacterium; contacting the antibody

with a label capable of being detected therebye identifying the presence of the antibody,

detecting the label, whereby the presence of Streptococcus mutans in the media is determined. The device for conducting these tests is a frame or support holding non-interactive material

capable of binding the sought antigens of interest (target) while permitting drainage of fluids.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtained by reference

to the following Description of the Preferred Embodiments and the accompaning drawings in

which like numerals in different figures represent the same structures or elements. The

representations in each of the figures is diagrammatic and no attempt is made to indicate actual

scales or precise ratios. Proportional relationships are shown as approximations.

Fig. la is an embodiment of the device for the method before developing the test.

Fig. lb is an embodiment of the device for the method showing the test fully

developed.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is directed to immunodiagnostic assays to identify cariogenic oral

bacteria. Polyclonal and/or monoclonal antibodies to Streptococcus mutans are used to

identify the caries. Polyclonal antibodies directed against putative oral pathogens can be

incorporated into an immunodiagnostic to diagnose active dental caries infections. The

antibodies provide a means of detecting antibodies for rapid clinical, chairside detection of oral

cariogenic microbial antigens. This rapid test method for determining the presence of

Streptococcus mutans in oral clinical specimens is fiilly developed and readable in under an

hour, usually about 5 minutes or less and the method and equipment is technically easy to use.

Within this invention the term rapid test is a test that can be developed in under an hour,

preferably in less than one-half hour. Most preferably, this test is fully readable in under

fifteen minutes from the removal of a plaque sample from the patient to the reading of the test. The presence of certain levels of Streptococcus mutans is used as a measuring rod to

indicate an active carious dental lesion. The test has potential for both whole-mouth screening

and site-specific applications.

Oral cariogenic infections pose a serious oral health problem. This invention will allow

the identification of patients that are at higest risk for developing dental caries; it will also permit the risk of individual carious lesions to be assessed. It is planned as a cornerstone of

an institutional caries risk assessment program. The value is that by knowing caries risk,

treatment resources can be directed in the most effective and efficient manner. The primary

advantage of this test method is that it can be performed and read in about 5 minutes compared

to 48 hours required for the earlier tests. The method is sensitive, specific and semi-

quantitative. The method can be used in a dental operatory with positive results obtained

while the patient is still in the chair. It allows rapid treatment decisions and can be used as

a dental caries screening device or to evaluate site-specific lesions.

In general, the antibodies, either polyclonal or monoclonal, are prepared in rabbits

although other animals can be used. Antibodies are naturally produced biomolecules which

react specifically with other foreign biomolecules called antigens. Antibodies are created by

immunizing an animal, preferably in this case rabbits, with killed bacteria that are associated

with human dental caries. A process such as described by Ebersole in U.S. Patent N^e

4,458,014 can be used to prepare the monoclonal or polyclonal antibodies. This immunization

causes the rabbits to produce antibodies that are directed against these bacteria (17). In

purified form, these antibodies are used as reagents to aid in the rapid detection of the bacteria

and early diagnosis of active dental caries in humans. In general the invention is a clinical diagnostic method using polyclonal antibodies

directly or indirectly to detect the presence of dental caries-related bacteria (Streptococcus

mutans) by a detectable label. The method comprises gathering a sample suspected of

containing an oral bacterium of interest (target) from a patient. The sample can be gathered

by any of the known techniques for gathering plaque and saliva samples. The sample is placed

on a substrate, preferably a flow through filter type device such as marketed by or a

device such as described by Oprandy in U.S. Patent N^a 5,039,493. Any oral bacterium

present on a non-inactive substrate is immobilized on the substrate and the other materials are

washed away. The substrate can be any of the commonly used substrates such as

nitrocellulose filter media, latex beads or any of the materials described by Oprandy. Once

the unkown sample is imobilized on the substrate, the sample is contacted with a previously

prepared antibody, either polyclonal antibodies (absorbed animal antisera) or monoclonal

antibodies, that are specific for the sought or target oral bacterium. The antibody is prepared

by known means as described by Ebersole supra or others. The antibody is contacted with a

label capable of being detected thereby e identifying the presence of the antibody. Any

detectable label can be used such as enzymes (e.g., alkaline phosphatase; peroxidase;

galactosidase; etc.) which react with their substrates to yield an insoluble end product.

Prefered labels are labels such as colloidal gold coupled to protein-A, protein-G, or some other

protein. Other detectable labels are such as fluorescent markers, radionuclides and others.

Once labeled, the presence of a target bacterium can be identified by detecting the label,

whereby the presence of Streptococcus mutans in the media is determined.

Alternative embodiments could involve the use of monoclonal antibodies to

Streptococcus mutans instead of polyclonal rabbit serum. Also, the use of colloidal gold or other labels such as enzymes or fluorochromes could be attached to several probes such as

protein A, protein G, goat anti-rabbit IgG, goat-anti-mouse IgG, and others.

Having described the invention, the following examples are given to illustrate specific

applications of the invention including the best mode now known to perform the invention.

These soecific examples are not intended to limit the scope of the invention described in this

application.

EXAMPLE

The test involves five simple and rapid steps:

a. The antigen (40 μg wet weight of Streptococcus mutans bacterial cells in 4 μl) is

spotted onto a flow-through filter device (Devaron, Princeton, NJ, 0.45 μm);

b. Nonspecific binding to the filter surface is blocked by adding 2 drops (80 μl) of 1 %

Bovine Serum Albumin in Phosphate-Buffered Saline with 0.1 % Tween-20;

c. Two drops (100 μl) of specific protein-A affinity purified and absorbed polyclonal

rabbit IgG to Streptococcus mutans serovars c, e, and f are then added;

d. The filter is washed and blocked just as in step 2; and

e. Five drops (200 μl) of either a protein-G (Sigma, P-1546; Protein G-5nm colloidal gold) or protein-A colloidal gold reagent in phosphate buffered saline with 12.5% glycerol and

0.25% bovine serum albumin (O.D. 1.5 at 522 nm) is added for color development. A pink

color developes in this test. The test is usually completed in five minutes or less. Color

changes which indicate the presence of Streptococcus mutans are evident in Fig. l b compared

to Fig. la.

New Zealand White rabbits were used for the development of the antibodies in the

serum. The New Zealand White rabbits are male, one year of age, and weigh approximately nine to ten pounds each. Three rabbits were used and were sufficient to provide the quantity

of antibody required against the bacteria of interest. The rationale for using these animals is

well established. Rabbits are widely used in production of antisera because of their ability to

respond to a variety of antigens and the large volume of available serum. The serological

production of antibodies in a rabbit system is a common and well documented procedure.

Established protocols are listed in the scientific literature. The immunization protocol

recommended by Ribi Immunochem Research, Inc. was used for the present example.

Antibodies to specific Streptococcus mutans serovars a-g (c, e, and f used in the present

prototype) are not commercially available.

a. Immunization: The rabbits were challenged with formalin-fixed and washed

Streptococcus mutans (strains ATCC 25175/NCTC 10449; LM-7; and OMZ-175) microbial

immunogens suspended in Monophosphoryl Lipid A (MPL) plus Trehalose Dimycolate (TDM)

Ribi Adjuvant System. A 50 mg wet weight (consisting of an equal mix of all three strains) suspension of the bacterial antigens was harvested by centrifugation, washed with sterile

physiological saline and reconstituted to 10 mg/ml in sterile saline and finally suspended in the

MPL + TDM adjuvant according to the manufacturer's instructions. A 1 ml dose/animal was

administered under aseptic technique as follows: 0.30ml intradermal, 0.40 ml intramuscular,

0.10 ml subcutaneous, and 0.20 ml intraperitoneal. The booster injection will be given

identically on day 21 or day 28.

b. Test bleeding. After 10 days from the booster injection, blood from a marginal

vein of the ear was collected to verify the antibody titer. Approximately 1-5 mis of whole

blood were collected from each rabbit. c. Terminal blood collection. Acceptable titered blood is collected by cardiac puncture

and the serum separated.

Semm was centrifuged at 10,000 x g and the supernatant was preserved by the addition of

0.1% sodium azide.

d. Absorption and Affinity purification of Antisera. The antisemm was made specific for

Streptococcus mutans serovars c, e, and f absorption with washed microbial antigens. First

the antisemm was affimty purified by using a pleated capsule recombinant protein A affinity

device (MASS, Nygene corp., Yonkers, NY). This membrane affinity purification results in

relatively pure IgG. For each 1 ml of rabbit IgG solution, 100 mg wet weight of formalin-

fixed washed whole cells of Streptococcus gordonii ATCC 10558 was mixed and incubated

with shaking at 37 °C for 1 hour followed by incubation for 12 hours at 4°C. The absorbed

antisera is the centrifuged at 16,000 x g for 60 min. The procedure was repeated using the

following microbial antigens: Streptococcus mutans isolates AHT, BHT, and Streptococcus

sobrinus isolates 6715-13 and SL-1. This reagent is the specific anti-Streptococcus mutans

serovars c, e, and f reagent used in the present prototype test system. This forms the antibodies that are stored for later use in the rapid assay.

The test does not have to be conducted in the particular order between immobilizing

the antibody and antigen. In a prefered commercial embodiment, the known antibody is

immobilized on a substrate, prefereably nitrocellulose media to form a test strip. The test strip

is then packaged, preferably with a vial of label or indicator, until needed. When needed, the

strip is removed from packaging, a sample is placed on the antibody spot and the sample

washed so any antigen interacts with the antibody. Then an indicator interactive with the

antigen is applied. When the indicator is gold, a color will develop as shown in Figure 1. Advantages and New Features

The primary advantage of this test method is that it can be performed and read in about

5 minutes compared to 48 hours required for the earlier tests. The method is sensitive,

specific and semi-quantitative. The method can be used in a dental operatory with positive

results obtained while the patient is still in the chair. It allows rapid treatment decisions and

can be used as a dental caries screening device or to evaluate site-specific lesions.

Obviously, many modifications and variations of the present invention are possible in

light of the above teachings. It is therefore to be understood that, within the scope of the

appended claims, the invention may be practiced otherwise than as specifically described.

Claims

What is claimed is:

Claim 1. An immunodiagnostic assay test strip for rapidly detecting the presence of

Streptococcus mutans (especially serovars c, e, and f) in human dental plaque and/or

radiographic lesions comprising:

a substrate capable of immobilizing an antigen or antibody,

an antibody for a specific oral bacteria imobilized on the substrate, and

an indicator capable of attaching to an antigen interactive with the immobilized

antibody.

Claim 2. The assay of claim 1 wherein the detectable label is colloidal gold coupled to

protein-A, protein-G, or some other protein.

Claim 3. An assay method for detecting oral bacterium comprising gathering a sample

suspected of containing an oral bacterium of interest, immobilizing any oral bacterium present

on a non-inactive substrate, contacting the sample with an antibody selected from the gropup

consisting of polyclonal antibodies and monoclonal antibodies that are specific for the target

oral bacterium; contacting the antibody with a label capable of being detected therebye

identifying the presence of the antibody, detecting the label, whereby the presence of oral

bacterium in the media is determined.

Claim 4. A method for detecting Streptococcus mutans (especially serovars c, e, and f) using:

a) either polyclonal antibodies (absorbed animal antisera) or monoclonal antibodies that

are specific for this oral bacterium;

b) contacting a reactive antibody directly or indirectly with a detectable label using

media suspected to contain Streptococcus mutans; c) detecting the label, whereby the presence of Streptococcus mutans in the media is

determined.

Claim 5. A method according to claim 4, wherein the detectable label is colloidal gold.

Claim 6. A method according to claim 4 wherein the detectable label is an enzyme.

Claim 7. A method according to claim 4 wherein the detectable label is a fluorescent marker.

Claim 8. A method according to claim 4 wherein the detectable label is a radionuciide.