WO2016196323A1

WO2016196323A1 - Serodiagnosis of melioidosis and glanders using polysaccharides

Info

Publication number: WO2016196323A1
Application number: PCT/US2016/034732
Authority: WO
Inventors: Paul James BRETT; Mary Nicole BURTNICK
Original assignee: University of South Alabama
Current assignee: University of South Alabama
Priority date: 2015-05-29
Filing date: 2016-05-27
Publication date: 2016-12-08
Anticipated expiration: 2017-11-29

Abstract

The present disclosure generally pertains to methods for the serodiagnosis of melioidosis and glanders in a subject. In one exemplary embodiment, the method for the serodiagnosis of melioidosis and glanders in a subject comprises the steps of: obtaining a sample from a subject; incubating the sample from the subject with at least one Burkholderia polysaccharide; and diagnosing the subject with glanders or melioidosis when the formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides is detected.The at least one purified Burkholderia polysaccharide may comprise O-polysaccharide, capsular polysaccharide, or any combination thereof.

Description

SERODIAGNOSIS OF MELIOIDOSIS AND GLANDERS USING

POLYSACCHARIDES

Cross Reference to Related Application

[0001] This application claims priority to U. S. Provisional Patent Application No.

62/230, 158, entitled "Serodiagnosis of Melioidosis and Glanders Using Polysaccharides" and filed on May 29, 2015, which is incorporated herein by reference.

Background of the Invention

[0002] Melioidosis is a potentially fatal disease caused by the environmental Gram- negative bacterium Burkholderia pseudomallei. The disease is endemic in tropical countries and continues to be a significant public health concern with increasing numbers of cases reported over the past century in many countries across the world. Management of melioidosis in patients can be challenging due to the requirement of specific antibiotic treatment with ceftazidime or a carbapenem. In northeast Thailand, for example, the mortality rate of such patients is 40%, and melioidosis is the third most common cause of death from infectious diseases after acquired- immunodeficiency syndrome (AIDS) and tuberculosis. B. pseudomallei typically infects people following exposure to contaminated soil and water by inoculation, inhalation or ingestion. The clinical spectrum of melioidosis is diverse, consisting of acute fulminant septicemia, subacute illness, chronic infection and subclinical disease. Melioidosis is often associated with a prolonged fever and bacteremia, and it can involve multiple organ infections. In acute forms, death can occur within 24-48 hours of the onset of symptoms.

[0003] The standard diagnostic method for melioidosis is bacterial culture followed by biochemical identification. The culture method is specific, but it is limited by low sensitivity and takes several days before a result is available. The current standard serology test for melioidosis is an indirect haemagglutination assay (IHA), which is widely used due to its simplicity to perform. Unfortunately, the IHA has been reported to be unreliable in many studies, and it has a low specificity and limited shelf-life, because it is prepared using sheep red blood cells sensitized with crude B. pseudomallei antigen. In addition, since the IHA is prepared from viable bacterial cultures, the preparation of the assays in countries where B. pseudomallei is categorized as a Biological Select Agent or Toxin ("select agent") or the equivalent thereof is neither desirable nor practical in light of the restrictions imposed on such select agents. Further, the IHA is poorly standardized, because different laboratories use different strains for antigen preparations.

[0004] To address these issues, Applicants have developed agglutination assays for the rapid serodiagnosis of melioidosis based upon surface exposed carbohydrate antigens expressed by B. pseudomallei. The O-polysaccharide (OPS) component of lipopolysaccharide (LPS), and the 6-deoxyheptan capsular polysaccharide (CPS) were selected as potential candidate antigens for serodiagnostic tests because they are highly conserved across B. pseudomallei strains, but are structurally different from other bacterial pathogens. In addition, OPS and CPS are well-characterized polysaccharide antigens, and both OPS-specific and CPS-specific antibodies can be detected in melioidosis patient serum.

Summary of the Invention

[0005] In certain embodiments, the present disclosure pertains to a method for detecting, in a sample from a subject, antibodies to Burkholderia polysaccharides, the method comprising the steps of: obtaining a sample from a subject; incubating the sample with at least one Burkholderia polysaccharide; and detecting the formation of at least one antibody-polysaccharide complex comprising at least one purified Burkholderia polysaccharide, wherein formation of the at least one complex is substantially indicative of antibodies to at least one Burkholderia polysaccharide being present in the sample. In certain embodiments, the at least one polysaccharide is selected from the group consisting of B. pseudomallei, Burkholderia mallei, and Burkholderia thailandensis , or a near-neighbor species, or is chemically synthesized. In certain embodiments, the method comprises incubating a sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O-polysaccharide and at least one is a capsular polysaccharide. In certain embodiments, the at least one purified Burkholderia polysaccharide is conjugated to a ligand. In certain embodiments, the at least one purified Burkholderia polysaccharide is biotinylated. In certain embodiments, the at least one purified Burkholderia polysaccharide is covalently linked to a peptide or protein. In certain embodiments, the at least one purified polysaccharide is attached or immobilized to a solid support. In certain embodiments, the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.

In certain embodiments, the present disclosure pertains to a method for diagnosing glanders or melioidosis disease in a subject, the method comprising the steps of: obtaining a sample from a subject; incubating the sample from the subject with at least one Burkholderia polysaccharide; and diagnosing the subject with glanders or melioidosis when the formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides is detected. In certain embodiments, the method comprises incubating a sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O-polysaccharide and at least one is a capsular polysaccharide. In certain embodiments, the at least one purified Burkholderia polysaccharide is conjugated to a ligand. In certain embodiments, the at least one purified Burkholderia polysaccharide is biotinylated. In certain embodiments, the at least one purified Burkholderia polysaccharide is covalently linked to a peptide or protein. In certain embodiments, the at least one purified polysaccharide is attached or immobilized to a solid support. In certain embodiments, the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.

[0006] In certain embodiments, the present disclosure pertains to a kit comprising at least one Burkholderia polysaccharide. In certain embodiments, the at least one Burkholderia polysaccharide comprises a mixture of purified O-polysaccharide and capsular polysaccharide. In certain embodiments, the kit includes a labeling reagent capable of binding to an antibody that recognizes the at least one purified Burkholderia polysaccharide. In certain embodiments, the at least one purified Burkholderia polysaccharide is attached to a solid support. In certain embodiments, the solid support is selected from the group consisting of a resin, a membrane, a bead, or a microtiter plate well. In another embodiment, the labeling reagent is a protein conjugated to a detectable label. In another embodiment, the detectable label is utilized in combination with a solid support that is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.

Brief Description of the Figures

[0007] The disclosure can be better understood with reference to the following figures.

[0008] Fig. 1 depicts the agglutination of antigen-coated latex beads following incubation with antigen-specific immune serum.

[0009] Fig. 2 depicts the conjugation of chemically activated B. pseudomallei OPS and CPS antigens to amine-derivatized latex beads.

[0010] Fig. 3 depicts IHA titers of melioidosis patients (A), healthy Thai donors (B), and healthy U. S. donors (C).

[0011] Fig. 4 depicts positive and negative results of OPS-latex agglutination test (A) and CPS-latex agglutination test (B) of all serum samples compared to IHA titers.

Detailed Description

The present disclosure generally pertains to methods for the serodiagnosis of melioidosis and glanders in a subject. In one exemplary embodiment, the method for the serodiagnosis of melioidosis and glanders in a subject comprises the steps of: obtaining a sample from a subject; incubating the sample from the subject with at least one Burkholderia polysaccharide; and diagnosing the subject with glanders or melioidosis when the formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides is detected. The at least one purified Burkholderia polysaccharide may comprise O-polysaccharide, capsular polysaccharide, or any combination thereof.

As used herein, "Burkholderia polysaccharide" means a polysaccharide selected from Burkholderia pseudomallei, Burkholderia mallei, Burkholderia thailandensis , or a near-neighbor species. A "Burkholderia polysaccharide" may also be chemically synthesized.

[0012] As used herein, "detectable label" means a chemical compound that can be detected either directly or indirectly by visual or instrumental means. A detectable label may consist of a molecule that itself produces a signal that can be detected, such as a fluorescent, chemiluminescent or radioactive signal. Altematively, the signaling label may comprise a molecule that requires reaction with another molecule to generate a signal that can be detected. Detectable labels also include compounds that can be detected visually, for example, colored dyes.

[0013] As used herein, "sample" means a biological sample collected from a subject.

Examples of a "sample" include, but are not limited to, whole blood, serum, plasma, saliva and other biological samples known in the art.

[0014] As used herein, the term "subject" means any and all organisms. "Subject" may refer to a human or any other animal. "Subject" may also refer to a fetus. Burkholderia polysaccharide can be naturally occurring or synthetic. Suitable methods of purification of a Burkholderia polysaccharide include, but are not limited to, chemical and detergent extraction of whole Burkholderia cells pellets and precipitation from Burkholderia cell culture supernatants, as known to those skilled in the art.

In certain embodiments, Burkholderia polysaccharide comprises at least one purified Burkholderia polysaccharide conjugated to a ligand. The benefit of ligand conjugation is the enablement of immobilization or attachment to a solid support. Suitable methods of activation for conjugation include, but are not limited to, oxidation and cyanylation. Ligands suitable for Burkholderia polysaccharide are known to those skilled in the art.

In certain embodiments, Burkholderia polysaccharide comprises at least one purified, biotinylated Burkholderia polysaccharide. Biotinylation enables immobilization or attachment of a Burkholderia polysaccharide to a solid support. Suitable methods of activation for biotinylation include, but are not limited to, oxidation and cyanylation.

In certain embodiments, Burkholderia polysaccharide comprises at least one purified Burkholderia polysaccharide that is covalently linked to a peptide or protein. Covalent linking enables immobilization or attachment of a Burkholderia polysaccharide to a solid support. Suitable methods of activation for covalent linking include, but are not limited to, oxidation and cyanylation. Suitable peptides are known to those skilled in the art. Suitable proteins include, but are not limited to, bovine serum albumin, OVA, bacterial proteins or toxoids, mammalian proteins, and plant proteins.

In certain embodiments, Burkholderia polysaccharide comprises at least one purified Burkholderia polysaccharide that is attached or immobilized to a solid support. Attachment or immobilization of a Burkholderia polysaccharide to a solid support may facilitate use in a detection assay. Suitable methods of immobilization are known in the art. In certain embodiments, covalent linking is used for immobilization. In other embodiments, non-covalent linking is used for immobilization. Suitable operating conditions for attachment or immobilization of a Burkholderia polysaccharide are known in the art. Suitable types of solid supports include, but are not limited to, a resin, a membrane, a bead, and a microtiter plate well. Suitable resins include, but are not limited to, agarose, acrylamide, and sepharose. Suitable membranes include, but are not limited to, nitrocellulose and polyvinylidene fluoride. Suitable beads include, but are not limited to, latex, magnetic, and polylactic-co- glycolic acid.

[0020] In certain embodiments, the present disclosure pertains to a method for detecting, in a sample from a subject, antibodies to Burkholderia polysaccharides, the method comprising the steps of: obtaining a sample from a subject; incubating the sample with at least one Burkholderia polysaccharide; and detecting the formation of at least one antibody-polysaccharide complex comprising at least one purified Burkholderia polysaccharide, wherein formation of the at least one complex is substantially indicative of antibodies to at least one Burkholderia polysaccharide being present in the sample. Suitable methods for detecting such complex formation include agglutination, colorimetric development, chemiluminescence, fluorescence and other techniques known in the art. In certain embodiments, the at least one polysaccharide is selected from the group consisting of B. pseudomallei , Burkholderia mallei, and Burkholderia thailandensis , or a near-neighbor species, or is chemically synthesized. In certain embodiments, the method comprises incubating the sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O-polysaccharide and at least one is a capsular polysaccharide. In certain embodiments, the at least one purified Burkholderia polysaccharide is conjugated to a ligand. In certain embodiments, the at least one purified Burkholderia polysaccharide is biotinylated. In certain embodiments, the at least one purified Burkholderia polysaccharide is covalently linked to a peptide or protein. In certain embodiments, the at least one purified polysaccharide is attached or immobilized to a solid support. In certain embodiments, the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well. Figure 1 depicts an embodiment of the presently disclosed method, wherein at least one latex bead acts as a solid support for at least one purified Burkholderia polysaccharide, which, in the presence of antibodies specific for the least one Burkholderia polysaccharide, forms an antibody-bead complex.

[0021] In certain embodiments, the present disclosure pertains to a method for diagnosing glanders or melioidosis disease in a subject, the method comprising the steps of: obtaining a sample from a subject; incubating the sample from the subject with at least one Burkholderia polysaccharide; and detecting formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides, wherein formation of the at least one complex is substantially indicative of the subject having been exposed to B. pseudomallei, B. mallei, B. thailandensis or a near-neighbor species. Suitable methods for detecting complex formation include agglutination, colorimetric development, chemiluminescence, fluorescence and other techniques known in the art. In certain embodiments, the method comprises incubating a sample with at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O- polysaccharide and at least one is a capsular polysaccharide. In certain embodiments, the at least one purified Burkholderia polysaccharide is conjugated to a ligand. In certain embodiments, the at least one purified Burkholderia polysaccharide is biotinylated. In certain embodiments, the at least one purified Burkholderia polysaccharide is covalently linked to a peptide or protein. In certain embodiments, the at least one purified polysaccharide is attached or immobilized to a solid support. In certain embodiments, the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.

[0022] In certain embodiments, the present disclosure pertains to a kit comprising at least one purified Burkholderia polysaccharide and a labeling reagent capable of binding to an antibody that recognizes one or more purified Burkholderia polysaccharides. In certain embodiments, the at least one polysaccharide is selected from the group consisting of B. pseudomallei, B. mallei, and B. thailandensis, a near- neighbor species, or are chemically synthesized. In certain embodiments, the at least one purified Burkholderia polysaccharide is immobilized or attached to a solid support. The solid support can be a resin, a membrane, a bead, a microtiter plate well or other solid support known in the art. In certain embodiments, the labeling reagent is a protein conjugated to a detectable label.

Examples

Materials and Methods

Serum samples

[0023] Three sets of human serum samples were used. The first set consisted of 143 sera from culture-confirmed B. pseudomallei infected patients who were admitted to Sappasithiprasong hospital, Ubon Ratchathani, northeast Thailand. This serum was left over from a previous study. The second set was 199 serum samples obtained from healthy donors from the same area in northeast Thailand. The last set was 90 serum samples obtained from healthy U.S. donors (Innovative Research, Novi, MI, USA). All serum samples were stored at -80°C. Freeze and thaw was kept to a minimum. All serum samples were used anonymously for diagnostic development. The study was approved by Ethics Committee of Faculty of Tropical Medicine, Mahidol University. Purification of B. pseudomallei OPS and CPS

[0024] Broth in 2 L baffled Erlenmeyer flasks was inoculated with the select agent excluded strains B. pseudomallei RR2808 (CPS mutant) or RR2683 (OPS mutant) and incubated overnight at 37°C with vigorous shaking. Cell pellets were obtained by centrifugation and extracted using a modified hot aqueous-phenol procedure. Purified OPS and CPS antigens were then obtained as previously described [Burtnick MN, et al. Front Cell Infect Microbiol 2: 108 (2012); Burtnick MN, et al. Front Cell Infect Microbiol 2: 148 (2012)].

Activation of latex beads with B. pseudomallei OPS and CPS

[0025] Purified OPS and CPS antigens were solubilized at 5 mg/ml in PBS pH 7.2 and added to small amber vials. To each ml of the OPS and CPS solutions, 6 mg (30 mM) of sodium meta-periodate was added. Once the crystals had dissolved, the reaction mixtures were incubated for 40 minutes at room temperature with stirring. To remove any excess oxidizing agent, the reaction mixtures were applied to 10 ml Zeba Desalt Spin Columns (Pierce) equilibrated with PBS and the eluates collected. To facilitate conjugation of the OPS and CPS to amine derivatized latex beads (1 μιτι; Life Technologies) (Figure 2), 1.6 ml of each polysaccharide solution (5mg/ml) was mixed with 8 ml of the latex beads (12.5 mg/ml in PBS) in 15 ml conical tubes. Following mixing, 10 μΐ of a 1 M sodium cyanoborohydride stock (in 10 mM NaOH) was added to each ml of the conjugation mixtures, and the reactions were incubated at room temperature for 24 hours with gentle mixing on a rotating platform. The latex beads were washed with PBS to remove any un-reacted polysaccharide then incubated overnight with 10 ml of Starting Block T20 (TBS; Pierce). The polysaccharide- modified beads were then washed twice with PBS, resuspended in 12 ml of PBS and stored at 4°C until required for use.

Indirect haemagglutination

[0026] IHA antigens prepared from two clinical B. pseudomallei isolates, strains 199a and 207a, were pooled and used to sensitize sheep red blood cells as described in Wuthiekanun V, et al. J Clin Microbiol 44:239 (2006). Before testing, the serum (50 μΐ) was inactivated at 56°C for 30 min, followed by pre-adsorption with 10% non- sensitized sheep red blood cells in PBS pH 7.2. 25 μΐ of 1% sensitized and non- sensitized cell controls were incubated with 50 μΐ of two-fold serial dilutions of each serum sample in 96-well plates as described in Wuthiekanun V, et al. Am J Trop Med Hyg 74: 1074 (2006). The results were read at the highest antibody titer that showed agglutination after incubating at room temperature for 2 hours. Positive IHA results were determined at a cut-off dilution > 1 : 160.

Latex agglutination tests

[0027] The OPS- and CPS-latex agglutination tests were initially evaluated for sensitivity and specificity using pooled serum from five melioidosis patients or five healthy Thai donors. To perform the assays, 10 μΐ of OPS-latex beads, CPS-latex beads, or uncoated latex beads were separately mixed with an equal volume of each serum sample on a glass slide and rotated at room temperature for 5 minutes. When conducting the assays, serum was used at a 1 : 10 dilution (in PBS) for the OPS-latex test and undiluted for the CPS-latex test and uncoated latex beads. The agglutination results were observed by eye against a black card and recorded as either positive (agglutination) or negative (no agglutination).

Statistical analysis

[0028] Statistical analyses were performed using Stata version 12 (StataCorp LP,

College Station, TX, USA). The McNemar test was used to compare the sensitivity and specificity between the different tests. The agreement between different tests was determined by Kappa analysis. The nonparametric test for trend of latex agglutination results was performed across the ordered groups of IHA titers. Differences were considered statistically significant if p-value < 0.05.

Results

Evaluation of polysaccharide-based latex agglutination tests and IHA titers

[0029] A diagnostic evaluation of the OPS-latex and CPS-latex agglutination assays was conducted, and the results were compared to the corresponding IHA titers of the samples tested. Two serum samples from the melioidosis group (N = 143) and eleven of the healthy Thai donors (N = 199) demonstrated agglutination with the uncoated latex beads (false positives) and were excluded from our analysis. Thus, the serum samples analyzed included 141 patients with culture-confirmed melioidosis, 188 healthy donors from Thailand and 90 healthy donors from the United States. The melioidosis patients and healthy Thai donors exhibited a wide range of IHA serum titers (Figure 3). The range for the melioidosis patients was 0 to > 1 : 10,240 (median 1 :320, IQR 1 :80-1 : 1,280) and for the healthy Thai donors was 0 to 1 :5,120 (median 1 :40, IQR 1 : 10-1 :320). In contrast, the IHA titers of the healthy U.S. donors were less than 1 :80 in all cases and ranged between 0 and 1 :40 (median 0, IQR 0).

The diagnostic sensitivity of CPS-latex was comparable to the IHA (69.5%; 98 of 141 patients for both, P =1.000). The sensitivity of the OPS-latex (84.4%; 119 of 141 patients) was significantly higher than the IHA (69.5%; 98 of 141 patients) (P = 0.001). When using serum from healthy Thai donors for analysis, the specificity of the CPS-latex (63.8%; 120 of 188 healthy donors) was comparable to the IHA (67.6%; 127 of 188 healthy donors) (P= 0.296). In contrast, the specificity of OPS-latex was significantly lower (56.9%; 107 of 188 healthy donors) than the IHA (P = 0.002). When evaluated with serum from healthy U.S. donors, all three tests were highly specific. The specificities of CPS-latex and OPS-latex were both 97.8% (88 of 90 healthy U.S. donors) and the IHA was 100%. Only two weakly positive reactions with either the CPS-latex (IHA titer 1 :20 for both sera) or OPS-latex (IHA titer 1 : 10 for both sera) were observed for the healthy U.S. donor samples.

IHA titers are in agreement with the polysaccharide-based latex agglutination tests

Kappa analysis of all samples showed that the results of OPS-latex and CPS- latex were in good agreement with the IHA results (agreement 80.7%, kappa 0.61 and P < 0.001 for OPS-latex and agreement 83.5%, kappa 0.65, P < 0.001 for CPS-latex). There were trends in OPS-latex and CPS-latex results across the ordered levels of IHA titers (P < 0.001 for both) (Figure 3). Interestingly, Applicants observed a prozone phenomenon (i.e., a false negative response) which showed false negative latex agglutination resulting from overwhelming antibody IHA titers at > 1 : 10,240 (1 of 13 patients for OPS-latex and 3 of 13 patients for CPS-latex) (Figure 3). Dilution of these serum samples by 2-4 fold resulted in positive agglutination reactions.

Both the OPS-latex and CPS-latex testing methods disclosed herein provide results that are comparable to the IHA test, which is currently the most commonly used serological test for melioidosis. Benefits of the Burkholderia polysaccharide-based latex agglutination tests disclosed herein include that they have a longer shelf-life than the IHA, are inexpensive, standardized and simple to perform. As such, the methods disclosed herein may be valuable diagnostic tests for rapidly detecting B. pseudomallei infections.

References to items in the singular should be understood to include items in the plural, and vice versa, unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive and conjunctive combinations of conjoined clauses, sentences, words, and the like, unless otherwise stated or clear from the context. Thus, the term "or" should generally be understood to mean "and/or" and so forth.

The various embodiments described herein are exemplary. Various other compositions for the kits and methods described herein are possible.

Claims

CLAIMS Now, therefore, the following is claimed:

1. A method for diagnosing glanders or melioidosis in a subject, comprising the steps of:

obtaining a sample from the subject;

incubating the sample from the subject with at least one purified Burkholderia polysaccharide; and

diagnosing the subject with glanders or melioidosis when the formation of at least one antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides is detected.

2. The method of claim 1, wherein the at least one Burkholderia polysaccharide is conjugated to a ligand.

3. The method of claim 1, wherein the at least one Burkholderia polysaccharide is biotinylated.

4. The method of claim 1, wherein the at least one Burkholderia polysaccharide is covalently linked to a peptide or protein.

5. The method of claim 1, wherein the at least one Burkholderia polysaccharide is immobilized to a solid support.

6. The method of claim 6, wherein the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.

7. The method of claim 6, wherein the solid support is a bead.

8. The method of claim 1, wherein the at least one purified Burkholderia polysaccharide comprises O-polysaccharide.

9. The method of claim 1, wherein the at least one purified Burkholderia polysaccharide comprises capsular polysaccharide.

10. The method of claim 1 , wherein the at least one purified Burkholderia polysaccharide comprises at least two purified Burkholderia polysaccharides, wherein at least one of the purified polysaccharides is O-polysaccharide and at least one is capsular polysaccharide.

11. A method for detecting antibodies to Burkholderia polysaccharides in a subject, comprising the steps of:

obtaining a sample from the subject;

detecting the formation of an antibody-polysaccharide complex comprising at least one of the purified Burkholderia polysaccharides, wherein formation of the antibody-polysaccharide complex is substantially indicative of antibodies to at least one Burkholderia polysaccharide being present in the sample.

12. The method of claim 11, wherein the at least one Burkholderia polysaccharide is conjugated to a ligand.

13. The method of claim 11, wherein the at least one Burkholderia polysaccharide is biotinylated.

14. The method of claim 11, wherein the at least one Burkholderia polysaccharide is covalently linked to a peptide or protein.

15. The method of claim 11, wherein the at least one Burkholderia polysaccharide is immobilized to a solid support.

16. The method of claim 15, wherein the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.

17. A kit comprising:

at least one purified Burkholderia polysaccharide; and

a labeling reagent capable of binding to an antibody that recognizes one or more purified Burkholderia polysaccharides.

18. The kit of claim 17, wherein each purified Burkholderia polysaccharide is attached to a solid support.

19. The kit of claim 18, wherein the solid support is selected from the group consisting of a resin, a membrane, a bead, and a microtiter plate well.

20. The kit of claim 17, wherein the labeling reagent is a protein conjugated to a detectable label.