Rapid immunoassay of anthrax protective antigen in vaccine cultures and bodily fluids by fluorescence polarization

The inventive subject matter relates to a competitive method for estimating the concentration in a sample of a Bacillus anthracis protein or antibody thereof selected from the group consisting of protective antigen (PA), lethal factor (LF) and edema factor (EF). The method may employ the use of Fluorescence Polarization, FLT or FRET. The competitive methods are capable of detecting a target protein within 5 minutes within the range of 0.1 to 10.0 nM. The methods provide for the rapid detection and quantitation of bacteria, bacterial antigen or antibody in culture media or broth of growing cultures of bacteria, including B. anthracis by fluorescent methods.

FIELD OF THE INVENTION

The inventive subject matter relates to a competitive fluorescence method for estimating the concentration in a sample ofBacillus anthracisprotein or specific antibody. The method contemplates the use of FLT, FRET or FP.

BACKGROUND OF INVENTION

Oral fluids have been increasingly recognized as acceptable alternatives to serum for use in diagnostic tests for certain hormones, drugs, antibodies and antigens. However, diagnostic assays utilizing saliva and other oral fluids appear as entries in the National Library of Medicine MEDLARS database with a frequency of only 1 in 30 and 1 in 100, respectively, when compared to entries in which blood serum is reported. Oral fluids are collected without pain, needle sticks, or religious and social prohibitions, and use of these involves minimal risk or exempt protocols for the use of human subjects. The Department of Defense (DoD) has made salivary diagnostics a Future Naval Capability (FNC), and both the Office of Naval Research (ONR) and the Military Infectious Disease Research Program (MIDRP) of the US Army have funded projects to investigate the use of oral fluids or saliva to diagnose disease or monitor immunization status. The military has had a long-standing interest in saliva with ONR compiling in 1960 a review of all saliva references in the literature from 1888-1957 (1). In 1965, the US Army Medical Research Development Command supported a contract that published one of the first reports of human salivary antibodies to indigenous bacteria (2). The United States Department of Agriculture (USDA) recently sponsored a symposium to advance non- or minimally-invasive technologies to monitor health and nutritional status in the Special Supplemental Nutrition Program for Women, Infants and Children (WIC) using saliva as a diagnostic tool (3, 4). These considerations and continuing improvements in standardization of collection methods make these body fluids the diagnostic media of choice in the 21stcentury.

Recent reviews of the clinical chemistry and microbiology of saliva (5), the roles of saliva in health and disease (6), in diagnosing periodontal disease (7), and as a diagnostic fluid (8) indicate the growing prominence this matrix plays in medical diagnostics (9, 10). Saliva (6) and oral fluid (11) are distinct biochemically. They generally reflect the serum pool, but neither is a passive ultrafiltrate (passive requires no direct ATP use) of serum as previously thought (12). The presence of mucins, the polyanionic glycoproteins that increase salivary viscosity, and oral flora have been largely responsible for the lack of popularity of oral fluids in clinical research. Saliva presents several challenges for diagnostics: limited reference values have been published and the standardization of sample collection has fallen behind serum (13). Standardized saliva (14) and oral fluid (15) collection devices have only recently become available and should contribute to further investigations. Antibody containing oral fluids, gingival crevicular fluid (GCF) and oral mucosal transudate (OMT) arise due to hydrostatic pressure of the capillaries and venules associated with the lingual or buccal epithelium. They offer less variation than saliva and the best alternative to serum for antibody detection (16). GCF is similar to serum in protein composition but is significantly lower in protein concentration, being about 3% of the protein levels in blood (17,18). GCF volume is about one percent of total saliva volume in the healthy mouth (19). GCF is obtained by inserting an absorbent paper into the pocket or sulcus of a tooth (between the tooth and gingiva) after clearing the supragingival plaque (20). Its medical and dental use has not been reviewed since the 1970's (21, 22). OMT is 3-4 fold higher in protein concentration than saliva based on the IgG obtained using this device and is collected by placing a thick pad against the buccal mucosal surface juxtaposed between the parotid duct and the gingival crest (11). A current OMT device uses a salt-impregnated pad that is subsequently treated to release the antibody-containing fluid and retain the glycoproteins on the pad yielding “oral fluid”. One such oral fluid collection device has been licensed by Abbott Laboratories and eliminates the necessity for venipuncture (23).

The oral cavity as an immunological entity has been reviewed with respect to oral diseases (24) and microbiology (19). The use of oral fluids in diagnostic immunology includes detection of HIV (25); measles, mumps and rubella (26); hepatitis A (27); B (28); and C (29);Helicobacter pylori(30); dengue (31); and Chagas' disease (32). The current review will focus on the use of fluorescence polarization (FP)-based tests to detect antibodies to anthrax vaccine and to tuberculosis exposure using saliva, GCF or OMT as a test fluid.

Currently excepted methods for the clinical evaluation of patients using serum and oral fluids include enzyme-linked immunosorbent assays (ELISA), agglutination or radioimmunoassays (RIA). However, FP has distinct advantages over previous methods. Because FP is conducted in a fluid environment and because polarization is a general property of fluorescent molecules, FP assays have the potential to be less susceptible to non-specific interactions occurring at the cell surface and to interferences present in non-homogeneous sample fluids. However, despite the clear advantages of FP in diagnostics, only a limited number of non-commercial laboratories have adopted FP for detection of antibody in serum samples (33-35). High throughput screening (HTS) laboratories, on the other hand prefer FP based assays as a component of an automatable system capable of real-time evaluation of samples that are robust, flexible, sensitive and consistent with historical data (36).

Fluid-based assays are highly adaptable to the direct evaluation of oral fluids, saliva and serum for exposure to infectious agents including HIV, measles, mumps, rubella, hepatitis,Helicobacter pylori, dengue disease (37-43). Salivary and oral fluid assays have been increasingly recognized as a better, non-invasive alternative to serum-based diagnostics in detecting certain hormone, drug, antibody and antigen detection (44, 45).

Infectious disease rates and immunization strategies are critical to public health as well as a component of military preparedness. An integral component of the U.S. immunization strategy is the production of effective vaccines against agents such asBacillus anthracis(anthrax) as well as other emerging organisms suchMycobacterium tuberculosisandM. bovis, the causative agents oftuberculosis(47).

Pathology due toB. anthracisinfection is primarily due to the release by the organism of “protective antigen” (PA) in association with lethal factor (LF) and edema factor (EF) (48). The complete DNA and protein sequence of PA has been published and its three-dimensional structure is known from x-ray crystallography (49). The characteristics and biological functions of the four domains of PA are also available permitting selection of epitopes within the domains based on antigenic properties (49-53). In animal studies, as well as studies of natural human infection, it was shown that individuals who survived an infection produced antibodies to PA suggesting its importance in protection (54).

Vaccination for anthrax occurs for all Department of Defense personnel deploying to at-risk areas of operation. Furthermore, research personnel working with infectious strains of anthrax are vaccination, prophylactically. The current human anthrax vaccine (adsorbed) (AVA) licensed in the United States contains aluminum hydroxide adjuvant and consists mostly of PA from an attenuated, non-encapsulated strain ofB. anthracis(55,56). Several recombinant PA experimental plasmids have been produced which could be important as a vaccine component against the disease.

The production of bacteria or their products for use in vaccines requires routine monitoring of bacterial growth to obtain the material in optimum quantities and in a form most suitable for eliciting immunity. Expression of specific proteins, depending on the organism, is to a great extent dependent on the phase of bacterial cell growth in culture and the density of the protein or organism. Routine monitoring of growing cultures is, therefore, important. Assays for bacterial growth monitoring, must be relatively accurate, sensitive and able to be conducted in near real-time. Furthermore, the assay must be able to operate in the face of turbid media containing interfering substances, not related to the target protein being measured.

Current methods available to monitor bacterial growth include optical density measurement, which gives an estimation of bacterial density but no direct measure of specific protein concentrations being produced. More accurate methods include Western blot; enzyme-linked immunosorbent assay (ELISA); dot blots; which is a form of ELISA and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). ELISA methods, however, are inadequate due to either low sensitivity or erroneous results caused by interference effects by media constituents. SDS-PAGE methods are inadequate because they do not give real time information such that a culture composition cannot be estimated with accuracy given the time span between culture SDS-PAGE and interpreting the SDS-PAGE results.

Fluorescent polarization (FP) technology is capable of rapid, real-time, sensitive evaluation of fluid phase antigens with high specificity (57, 58). FP based assays are predicated on the principle that polarized incident visible or ultraviolet light that illuminates a fluorochrome causes subsequent polarized fluorescence with emission at a longer wavelength. However, molecules in solution are capable of rotation. Therefore, polarized light striking a fluorescent molecule loses polarization due to rotation of the molecule. Solutions containing slower turning, large molecule-fluorochrome complexes tends to stay polarized longer verses situations where smaller labeled molecules are present. In order to accommodate molecules of different sizes (up to 107kDa molecular weight), different fluorochromes can be selected (59).

Combining fluorochrome-labeled antigen or peptide with antibody results in an increase in FP, as measured in arbitrary millipolarization (mP) units. The smaller the fluorescent antigen, the greater the increase in mP units that is measured upon binding to its corresponding antibody, since mP depends upon the partial specific volume (approximate molecular weight in solution) of the labeled substance. The dependence is non-linear but is describable in a Perrin equation (66).

FP antigen-antibody binding assays require only the mixing of fluorescent reagent (antigen) with the sample (containing antibody) in a liquid buffer. In a rapid diagnostic format, essentially two FP readings are necessary; a base-line reading and a reading after a specified time. The FP value increases as binding of antigen and antibody occurs in a direct binding assay. The difference in FP between a fluorescent antigen of 10 kDa initially and the fluorescent complex consisting of it and IgG, for example, results in a measurable association using less than saturating antibody concentrations (60).

A distinct advantage of FP technology is that assays can accommodate somewhat cloudy solutions, such as bacterial suspensions or variations in total fluorescence that are present in non-homogenous solutions, such as bacterial broth, urine or oral fluids. Furthermore, FP assays can be designed to accommodate significant variation in pH in fluid samples, such as in some media or in saliva or urine, by utilizing different pH-independent fluorochromes (61).

Currently, FP assays are in used to measure different types of binding reactions, to follow proteolytic reactions and to measure various enzymatic or receptor binding reactions (62, 63). In clinical settings, FP assays are used to measure the level of drugs, hormones or antibiotics in blood plasma (64). ELISA, RIA and immunoprecipitation assays are the most accepted methods for detection of antibody in serum samples (60, 65, 66). However, FP, because of its advantages over other detection methods is well-suited as a diagnostic tool for analyzing formerly difficult to evaluate samples such as oral fluids and saliva, in addition to serum, for the quantitative assessment of specific antibody, diagnostic markers, drugs, chemicals and infectious or biohazardous agents.

A further aspect of the invention is the detection and quantitation of protective antigen, lethal factor and edema factor fromBacillus anthracisin saliva, blood, oral fluids or other bodily fluids and tissues. Current vaccines toB. anthracisare directed against PA. Furthermore, because of the importance of lethal factor and edema factor in the pathology of anthrax, detection of these genes in body fluids or broth cultures is also important. Therefore, an inventive aspect is the specific, sensitive and rapid detection of PA in bodily fluids including saliva, and oral fluids and environmental samples.

SUMMARY OF THE INVENTION

Current methods for the monitoring of growing broth cultures of bacteria for vaccine production, including ELISA and SDS-PAGE, are inadequate. ELISA exhibits high levels of interference by media constituents leading to low fidelity of results and concomitant low sensitivity of detection. SDS-PAGE, like ELISA is extremely time consuming making the method unsuitable for routine monitoring. Therefore, a method that is sensitive, rapid and that gives predictable results is important in order to optimize production of bacterial vaccine components, including the vaccine components forB. anthracis, protective antigen (PA).

An object of the invention is an assay for the rapid detection and quantitation of bacteria in culture media or broth of growing cultures ofB. anthracisby competitive fluorescent polarization (FP), fluorescence lifttime (FLT) analysis or fluorescence resonance energy transfer (FRET). Another object of the invention is a rapid, real-time detection method for monitoring growing bacterial cultures or bodily fluids. A still further object is an assay capable of sensitive, rapid detection of bacterial media or bodily fluids without interference from sample constituents. A still further object is the real-time quantitation of protective antigen (PA) in growing cultures ofB. anthracis.A still further object is the quantitation of lethal factor (LF) in growing cultures ofB. anthracis.still further object is the quantitation of edema factor (EF) in growing cultures ofB. anthracis.A still further object is the detection of PA, LF and EF in bodily fluids, including saliva and oral fluids and in environmental samples.

These and other objects of the invention are accomplished by an assay employing fluorescent polarization in a competitive assay procedure.

DETAILED DESCRIPTION

Fluorescent polarization holds great promise as an assay method for frequent, routine, cost-effective means of obtaining quantitation of bacterial proteins in growing bacterial broth cultures. Fluorescent molecules emit polarized fluorescence when they absorb polarized light at a specific wavelength. However, inherent in molecules in solution is their tendency to rotate. When polarized light strikes the molecules in solution, the emitted light does not remain polarized because the molecule is rotating rapidly in solution. The rate of rotation, however, is dependent on the size of the molecule. Therefore, an antigen/antibody complex will have an inherently slower rate of rotation causing more of the polarized fluorescence to be emitted in the same plane as the incident light. The polarization-based readouts are less susceptible to environmental interferences, such as pH, cloudiness, and particles in the assay compartment than other light-based assays. FP is fundamentally and theoretically different from most color based photometric techniques and gains its analytical advantage by this intrinsic property of being independent of concentration. The current application utilizes FP technology in a competitive assay to quantitate bacterial protein concentration in growing bacterial broth cultures. The invention is designed to be capable of determining the concentration of antigen in fermentation broth.

An aspect of the invention is a competitive method for estimating the concentration ofBacillus anthracisproteins such as protective antigen (PA), lethal factor (LF) and edema factor (EF). The assay method incorporates the broad detection technologies of fluorescence polarization (FP), fluorescence life-time (FLT) and fluorescence resonance energy transfer (FRET). The general scheme of the assay includes the following steps:a. intermixing a sample, suspected of containing aB. anthracisPA, LF or EF with a specific antibody and a competitive reagent, suitable as a ligand for the specific antibody and labeled a fluorochrome;b. incubating the competitive reagent and specific antibody for 15 seconds to 5 minutes, depending on suspected concentration of the target antigen in the sample;c. detecting the binding interaction of PA, LF or EF and specific antibody.

Detection of specific agent would then be by either a change in fluorescence polarization or a change in fluorescence life-time, depending on whether the technology incorporated was FP or FLT, respectively. If the method incorporated FRET, then detection is by sensitized fluorescence of the acceptor or by quenching of donor fluorescence or by fluorescence depolarization. Furthermore, the competitive reagent used in the assay can be either native or recombinant PA, LF or EF or fragments thereof.

The assay method is generally adaptable to the detection of PA, LF or EF in bacterial culture media as well as in other bodily fluids including: saliva, oral rinse expectorant, oral fluid including oral mucosal transudate and gingival crevicular fluid, urine, sweat, tears, blood, serum, stool, gastric fluid, synovial fluid, phlegm, and other clinical and laboratory specimens and samples. The time of assay will depend on expected concentration in the sample. For bacterial cultures that has progressed near or past the logarithmic phase of its growth curve, it is estimated that 30 minutes or less will be required for incubation with specific antibody.

The results of test samples will be compared to results obtained from negative or positive control results, using either FP, FLT or FRET technology. Negative controls will contain the same fluorochrome-labeled competitive reagent as used in the test sample. A positive control can also contain the same fluorochrome-labeled competitive reagent as in the test sample but will be exposed to a known amount of said protein. The measured results of the test samples will then be compared to either or both of the negative or positive controls. It is estimated that the sensitivity of detection ofB. anthracisprotein will occur down to at least 5 μg/ml (6 nM) or less. The required time for the assay will be less than 5 minutes (see Prophetic Example 1).

In addition to detectionB. anthracisproteins in various samples types, a still further aspect of the invention is the detection of specific antibody to PA, LF or EF in bodily fluids by an assay method incorporation FP, FLT or FRET technology. The bodily fluids that are included as an aspect of the invention include: saliva, oral rinse expectorant, oral fluid including oral mucosal transudate and gingival crevicular fluid, urine, sweat, tears, blood, serum, stool, gastric fluid, synovial fluid, phlegm, and other clinical and laboratory specimens and samples.

The inventive subject matter for the detection of specific antibodies toB. anthracisproteins includes the steps:a. intermixing a sample, suspected of containing specific antibody with a competitive reagent labeled with a fluorochrome, capable of binding to the specific antibody;b. incubating the sample, containing specific antibody, and competitive reagent for 15 seconds to 5 minutes;c. detecting binding interaction between said protein and antibody.

Detection of specific agent would then be by either a change in fluorescence polarization or a change in fluorescence life-time, depending on whether the technology incorporated was FP or FLT, respectively. If the method incorporated FRET, then detection is by sensitized fluorescence of the acceptor or by quenching of donor fluorescence or by fluorescence depolarization.

The results of test samples will be compared to results obtained from negative or positive control results, using either FP, FLT or FRET technology. Negative controls will contain the same fluorochrome-labeled competitive reagent as used in the test sample. A positive control can also contain the same fluorochrome-labeled competitive reagent as in the test sample but will be exposed to a known amount of said protein. The measured results of the test samples will then be compared to either or both of the negative or positive controls.

For both detection ofB. anthracisantigen as well as antibody, specific forB. anthracisproteins, an aspect of the invention is that a fluorochrome concentration of 1 nM or less will yield a sample millipolarization increase or decrease of at least 10 mP. A further aspect is that the method will have a specificity of 96-99% for the detection of specific antibody or antigen in bodily fluids or culture.

Competitive FP Assay for Measuring Broth Protective Antigen, Lethal Factor and Endema Factor ofBacillus anthracis

Quantitative, real-time monitoring of growing bacterial cultures is necessary to ensure maximum production of important bacterial proteins in vaccine production.Bacillus anthracisis of particular importance due to its potential public health and military focus as a potential bioterrorism weapon. Monitoring of growing cultures ofB. anthracisis conducted by the following steps:a) Add 10 μl of labeled competitor peptide (e.g. FITC labeled PA, lethal factor or edema factor) in 1 ml buffer;b) Read blank;c) Add 20 μl of monoclonal antibody to target protein in said 1 ml buffer;d) Incubate for 2 minutes;e) Read the FP of the tracer;f) Add 10 μl of culture broth to said 1 ml buffer in step d);g) Read decrease in mP. The decrease in mP is proportional to the PA concentration.

Culture broths ofB. anthraciscontain protective antigen concentrations up to 5 ug/ml. Therefore the assayable range of protective antigen required for FP assay is similar to that for ELISA assay. Using standard sandwich ELISA techniques, recombinant PA reports approximately 0.01 OD per 1 ng PA at 405 nm using horse radish peroxidase substrate conjugated antibodies in a range of 1.5-200 ng protective antigen. An example standard ELISA was used to estimate protective antigen in serial dilutions of buffer from 25 nM (2 ug/ml) down to 0.2 nM (16 ng/ml).

Similar results are expected in competitive fluorescence polarization as immunoassay techniques (seeFIG. 1). Combining an fluorescent dye-labeled protective antigen probe at about 1 nM (labeled PA probe) and a monoclonal (MAb) or polyclonal (PAb) antibody at an adequate dilution (usually between 1:25-1:250K) would report numbers around 200 mP (left side of equation and left side of graph as shown inFIG. 1). Culture broth would be added as unlabeled competitor (broth PA) in a range of 0.01-0.1 ml depending on how long the culture had grown causing dissociation of the labeled probe from the antibody. Dissociation of the labeled probe from the antibody in the presence of increasing amounts of broth PA results in increasing amounts of free labeled probe and decreasing mP readings that are proportional to the concentration of protective antigen in the culture broth. (Right side of the equation and right side of the graph). At full growth, the assay mixture would require 0.01 ml broth (5 ug/ml protective antigen, about 6 nM) while partial growth would require proportionately more broth up to 0.1 ml to estimate a similar concentration. Other optional reagents may be used to suppress intrinsic culture broth fluorescence.

Determining the Concentration of Recombinant or Native Protective Antigen (PA) ofBacillus anthracisAntigen in Fermentation Broth

Overlapping 15 and 25 mer peptide sequences were synthesized from the amino acid region 1-764 of full-length PA using Fmoc (9-fluorenylmethoxycarbonyl) chemistry including a final step to attach an N-terminal 6-carboxyfluorescein (FAM). Enzymatic digests of fluorescein-6-isothiocyanate (FITC isomer II) labeled full length PA were made with trypsin, pepsin and chymotrypsin. These peptides were screened for specific antibody binding by immune sera. Using Western Blot, ELISA and dot-blot analysis, a 10-mer portion of domain 4 of the COOH region of PA was suitable as an FP probe, a 5-6 kDa portion of domain 3 and domain 4 was suitable as an FP probe, a 10 kDa portion of domain 3 and 4 was suitable as a FP probe, a 5-6 kDa portion of domain 1 and 2 was suitable as a FP probe, and a 10 kDa portion of domain 1 and 2 was suitable as a FP probe

Fermentation broth cultures ofBacillus anthracis(vaccine strain V770-NP1-R) containing PA were produced. Culture media is then mixed with antibody and competitive probe. Incubation times was less than 30 seconds for cultures nearing the stationary phase of growth and for less than 4 minutes for newly seeded cultures (and therefore low concentration of PA). At 1 nM probe concentration, the blank subtracted fluorescence polarization reading is determined to be quantifiable if mP decreased by at least 10 mP.

REFERENCES

Having described the invention, one of skill in the art will appreciate in the appended claims that 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.