Systems and methods for pathogen detection

Abstract

Described herein are systems, kits, and methods for detecting pathogens.

Description

Attorney Docket No.10620-163W01SYSTEMS AND METHODS FOR PATHOGEN DETECTIONCROSS REFERENCE

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 749,140, filed January 24, 2025, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Vector-borne pathogens (e.g., bacteria, protozoa) are transmitted by a variety of vectors, such as ticks and fleas. These pathogens cause or are associated with different diseases and conditions in humans and animals. Currently, diagnosis of some diseases and conditions associated with vector-borne pathogens are typically based on serological assays designed to detect an antibody produced by a subject in response to a pathogen. Despite molecular and biotechnological advances, serological assays employing immunofluorescence antibody (IFA), Western blotting (WB) and enzyme-linked-immunosorbent-assay (ELISA) technologies have encountered diagnostic limitations, primarily due to poor sensitivity. Therefore, there is a need to develop rapid and reliable serological assays for detecting, in a subject sample, an antibody response to a pathogen.SUMMARY

[0003] Described herein is a composition comprising two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-p (rATP-p), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5). In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. In some embodiments, the two or more recombinant pathogen polypeptides comprise immunodominant amino acid sequences. In some embodiments, the two or more immunodominant amino acid sequences are selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity,, hydrophilicity prediction, or a combination thereof. In some embodiments, the two or more recombinant pathogen polypeptides are attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any two orAttorney Docket No.10620-163W01more of SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the composition comprises recombinant ATP-b and recombinant GroEL.

[0004] Described herein is a system comprising the composition disclosed herein and an immunoglobulin binding agent, wherein the immunoglobin binding agent binds to an antibody, wherein the antibody binds to any one of the recombinant ATP-p (rATP-p), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5). In some embodiments, the immunoglobulin binding agent binds to a IgG. In some embodiments, the IgG comprises IgGl, IgG2, IgG3, or IgG4. In some embodiments, the system comprises a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding between the antibody and the two or more recombinant pathogen polypeptides. In some embodiments, the signal moiety is complexed with the immunoglobulin binding agent. In some embodiments, the signal moiety comprises an enzyme. In some embodiments, the enzyme comprises a horseradish peroxidase (HRP). In some embodiments, the signal moiety comprises a fluorescent molecule. In some embodiments, the antibody comprises an endogenous antibody. In some embodiments, the antibody binds to two or more of the recombinant pathogen polypeptides. In some embodiments, the antibody is produced in response to an infection from a pathogen. In some embodiments, the antibody is produced in response to vaccination against a pathogen. In some embodiments, the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinant pathogen polypeptides indicates an infection from a pathogen in the subject. In some embodiments, the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinant pathogen polypeptides indicates vaccination against a pathogen in the subject. In some embodiments, the pathogen comprises a Bartonella species. In some embodiments, the system comprises a therapeutic regimen for treatment against the infection from the pathogen or a vaccine for protection against the pathogen. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity of detecting the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity compared to the second specificity is increased by at least 5%, atAttorney Docket No.10620-163W01least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity of detecting the infection in the subject compared to a second sensitivity of detecting the infection in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the specificity of detecting the vaccination in the subject compared to a second specificity of detecting the vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the sensitivity of detecting infected versus vaccinated subjects. In some embodiments, the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, the antibody comprises a canine antibody. In some embodiments, the antibody consists of the canine antibody. In some embodiments, the antibody comprises a human antibody. In some embodiments, the antibody consists of the human antibody.

[0005] Described herein is a kit comprising the composition disclosed herein, an immunoglobulin binding agent; and an instruction manual,

[0006] Described herein is a method comprising capturing an antibody in a sample derived from a subject by contacting the sample with the composition disclosed herein, wherein the antibody binds to any one of the recombinant ATP-b (rATP-b), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5).

[0007] Described herein is a method comprising contacting a sample derived from a subject with the composition disclosed herein and detecting binding between an antibody in the sample and the two or more recombinant pathogen polypeptides.Attorney Docket No.10620-163W01

[0008] Described herein, in some aspects, is a system comprising: two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); and an immunoglobulin binding agent, wherein the immunoglobin binding agent binds to an antibody, wherein the antibody binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5). In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. In some embodiments, the two or more recombinant pathogen polypeptides comprise immunodominant amino acid sequences. In some embodiments, the two or more immunodominant amino acid sequences are selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, hydrophilicity prediction, or a combination thereof. In some embodiments, the two or more recombinant pathogen polypeptides are attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the immunoglobulin binding agent binds to a IgG. In some embodiments, the IgG comprises IgGl, IgG2, IgG3, or IgG4. In some embodiments, the system further comprises a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding between the antibody and the two or more recombinant pathogen polypeptides. In some embodiments, the signal moiety is complexed with the immunoglobulin binding agent. In some embodiments, the signal moiety comprises an enzyme. In some embodiments, the enzyme comprises a horseradish peroxidase (HRP). In some embodiments, the signal moiety comprises a fluorescent molecule. In some embodiments, the antibody comprises an endogenous antibody. In some embodiments, the antibody binds to two or more of the recombinant pathogen polypeptides. In some embodiments, the antibody is produced in response to an infection from a pathogen. In some embodiments, the antibody is produced in response to vaccination against a pathogen. In some embodiments, the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinantAttorney Docket No.10620-163W01pathogen polypeptides indicates an infection from a pathogen in the subject. In some embodiments, the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinant pathogen polypeptides indicates vaccination against a pathogen in the subject. In some embodiments, the pathogen comprises a Bartonella species. In some embodiments, the system further comprises a therapeutic regimen for treatment against the infection from the pathogen or a vaccine for protection against the pathogen. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity of detecting the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity of detecting the infection in the subject compared to a second sensitivity of detecting the infection in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the specificity of detecting the vaccination in the subject compared to a second specificity of detecting the vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the sensitivity of detecting infected versus vaccinated subjects. In some embodiments, the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, the antibody comprises a canine antibody. In some embodiments, the antibody consists of the canine antibody. In some embodiments, the antibody comprises a human antibody. In some embodiments, the antibody consists of the human antibody.Attorney Docket No.10620-163W01

[0009] Described herein, in some aspects, is a kit comprising: two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LeniA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); an immunoglobulin binding agent; and an instruction manual. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more immunodominant amino acid sequences. In some embodiments, the two or more recombinant pathogen polypeptides are attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species polypeptides. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15 In some embodiments, the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the kit further comprises a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding among: an antibody; the two or more recombinant pathogen polypeptides; and the immunoglobulin binding agent. In some embodiments, the signal moiety is complexed with the immunoglobulin binding agent. In some embodiments, the signal moiety comprises an enzyme. In some embodiments, the enzyme comprises a horseradish peroxidase (HRP) In some embodiments, the signal moiety comprises a fluorescent molecule. In some embodiments, the kit further comprises a therapeutic regimen for treating an infection from a pathogen or a vaccine for protection against the pathogen. In some embodiments, the pathogen comprises a Bartonella species.

[0010] Described herein, in some aspects, is a method comprising: capturing an antibody in a sample obtained from a subject by contacting the sample with two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5), wherein the antibody binds to binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5). Also describedAttorney Docket No.10620-163W01herein, in some aspects, is a method comprising: contacting a sample obtained from a subject with two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); and detecting binding between an antibody in the sample and the two or more recombinant pathogen polypeptides. In some embodiments, the method further comprises binding the antibody with an immunoglobulin binding agent. In some embodiments, the immunoglobulin binding agent binds to an IgG. In some embodiments, the IgG comprises IgGl, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobin binding agent is complexed with a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of a binding between the antibody and the two or more recombinant pathogen polypeptides. In some embodiments, the signal moiety comprises an enzyme. In some embodiments, the enzyme comprises a horseradish peroxidase (HRP). In some embodiments, the signal moiety comprises a fluorescent molecule. In some embodiments, the antibody comprises an endogenous antibody. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more immunodominant amino acid sequences. In some embodiments, the two or more immunodominant amino acid sequences are selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, hydrophilicity prediction, or a combination thereof. In some embodiments, the two or more recombinant pathogen polypeptides are attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species polypeptides. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the antibody is produced in response to an infection from a pathogen in the subject. In some embodiments, detecting the antibody in the sample binding to the two or more recombinant pathogen polypeptides indicates the infection from the pathogen in the subject. In some embodiments, the antibody is produced in response to vaccination against a pathogen in the subject. In some embodiments, detecting the antibody in the sample binding to the two or more recombinant pathogen polypeptides indicates vaccinationAttorney Docket No.10620-163W01against the pathogen in the subject. In some embodiments, the pathogen comprises a. Bartonella species. In some embodiments, the method further comprises treating the subject infected with the pathogen. In some embodiments, the method further comprises protecting the subject against the pathogen. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity for detecting the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity of detecting the infection in the subject compared to a second sensitivity of detecting the infection in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the vaccination in the subject compared to a second specificity for detecting the vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and two or more recombinant pathogen polypeptides increases the sensitivity of detecting infected versus vaccinated subjects. In some embodiments, the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%. In some embodiments, the antibody comprises a canine antibody. In some embodiments, the antibody consists of the canine antibody. In some embodiments, the antibody comprises a human antibody. In some embodiments, the antibody consists of the human antibody.INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, orAttorney Docket No.10620-163W01patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0013] FIG. 1 illustrates a flow diagram of an example assay workflow for detecting, in a sample obtained from a subject, an antibody response to a pathogen;

[0014] FIG. 2 illustrates a photograph of an example of an SDS-PAGE gel showing bands representing immunodominant proteins of 13, 17, 50, 56, and 150 KDa;

[0015] FIG. 3 illustrates a panel of plots showing predicted B-cell epitopes of Bartonella henselae F0F1 ATP synthase subunit beta, B. henselae GroEL and B. henselae LemA protein, respectively, obtained using the IEDB antibody epitope prediction tool;

[0016] FIG. 4 illustrates a panel of photographs showing an example of Coomassie stained SDS-PAGE and Western blot analysis of purified recombinant proteins rATP-P, rGroEL, rLemA, rSucB, and rVirB5;

[0017] FIG. 5 illustrates a panel of scatter plots of ELISA seroreactivity of recombinant proteins (rATP-P, rGroEL, rLemA, rSucB, and rVirB5, respectively) among Bartonella-infected and control dogs;

[0018] FIG. 6 illustrates a panel of plots showing receiver operating characteristic (ROC) curves with 95% Cis for ELISA seroreactivity of Bartonella recombinant proteins (rATP-P, rGroEL, rLemA, rSucB, and rVirB5, respectively) for dogs;

[0019] FIG. 7 illustrates a scatter plot and ROC curves for ELISA seroreactivity of Bartonella recombinant proteins rATP-P and rGroEL used in combination.

[0020] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will beAttorney Docket No.10620-163W01obtained by reference to the following detailed description that sets forth illustrative embodiments.DETAILED DESCRIPTION

[0021] Described herein are systems, methods, or kits for detecting, in a sample, an antibody response to a pathogen or a component of a pathogen. In some embodiments, the detecting of an antibody response to a pathogen comprises contacting a sample obtained from a subject with two or more recombinant pathogen polypeptides and detecting binding between an antibody in the subject sample and the two or more recombinant pathogen polypeptides.

[0022] In some embodiments, the systems, methods, or kits described herein may be used for detecting an antibody produced by a subject in response to an infection from a pathogen of interest. In some embodiments, the systems, methods, or kits described herein may be used for detecting an antibody produced by a subject in response to vaccination against a pathogen of interest.Composition for detecting an antibody response to a pathogen

[0023] The present disclosure provides a composition comprising two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5). In some embodiments, the composition comprises a recombinant ATP-P (rATP-p) or a recombinant GroEL (rGroEL). In some embodiments, the composition comprises a recombinant GroEL (rGroEL) or a recombinant LemA (rLemA). In some embodiments, the composition comprises a recombinant LemA (rLemA) or a recombinant SucB (rSucB). In some embodiments, the composition comprises a recombinant SucB (rSucB) and a recombinant VirB5 (rVirB5). In some embodiment, the composition comprises two, three, four, or five recombinant pathogen polypeptides. In some embodiment, the composition comprises two recombinant pathogen polypeptides. In some embodiment, the composition comprises three recombinant pathogen polypeptides. In some embodiment, the composition comprises four recombinant pathogen polypeptides. In some embodiment, the composition comprises five recombinant pathogen polypeptides.

[0024] In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. In some embodiments, the two or more recombinant pathogen polypeptides comprise immunodominant amino acid sequences.Attorney Docket No.10620-163W01

[0025] In some embodiments, the two or more immunodominant amino acid sequences may be selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, hydrophilicity' prediction, or a combination thereof. In some embodiments, the two or more recombinant pathogen polypeptides may be attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides.

[0026] In some embodiments, the two or more recombinant pathogen polypeptides are 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant pathogen polypeptides are about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15.System for detecting an antibody response to a pathogen

[0027] The disclosure provides a system for detecting an antibody response to a pathogen or a component of a pathogen of interest, the system comprising: (a) two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); and (b) an immunoglobulin binding agent, wherein (i) the immunoglobin binding agent binds to an antibody, and (ii) the antibody binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5); and (c) a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding between the antibody and the two or more recombinant pathogen polypeptides.

[0028] In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. The two or more cytoplasmic domains of the recombinant pathogen polypeptides may, for example, comprise immunodominant amino acid sequences selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, hydrophilicity' prediction, or a combination thereof. In some embodiments, the two or moreAttorney Docket No.10620-163W01recombinant pathogen polypeptides may be attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides.

[0029] In some embodiments, the two or more recombinant pathogen polypeptides are 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments, the two or more recombinant pathogen polypeptides are about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments, the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15.

[0030] In some embodiments, the immunoglobulin binding agent binds to an IgG antibody, wherein the IgG comprises IgGl, IgG2, IgG3, or IgG4.

[0031] In some embodiments, the antibody binding to the two or more recombinant pathogen polypeptides comprises an endogenous antibody produced by a subject in response to a pathogen or a component of the pathogen. In some embodiments, the antibody is produced in response to an infection from a pathogen. In this case, detecting binding between the antibody and the two or more recombinant pathogen polypeptides indicates an infection from a pathogen in the subject. In some embodiments, the antibody may be a canine antibody produced in response to an infection from a pathogen. In some embodiments, the antibody may be a human antibodyAttorney Docket No.10620-163W01produced in response to an infection from a pathogen. In some embodiments, the pathogen comprises a Bartonella species,

[0032] In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity of detecting the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity for detecting binding between the antibody and the two or more recombinant pathogen polypeptides compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%.

[0033] In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity for detecting the infection in the subject compared to a second sensitivity of detecting the infection in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the sensitivity for detecting binding between the antibody and the two or more recombinant pathogen polypeptides compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%.

[0034] In some embodiments, the antibody binding to the two or more recombinant pathogen polypeptides comprises an antibody produced by a subject in response to vaccination against a pathogen. In this case, antibody binding to the two or more recombinant pathogen polypeptides indicates vaccination against a pathogen in the subject. In some embodiments, the antibody may be a canine antibody produced in response to vaccination against a pathogen. In some embodiments, the antibody may be a human antibody produced in response to a vaccination against a pathogen. In some embodiments, the pathogen comprises. Bartonella species.

[0035] In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the vaccination in the subject compared to a second specificity of detecting the vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the specificity for detecting binding between the antibody and the two or more recombinant pathogen polypeptides compared to the second specificity isAttorney Docket No.10620-163W01increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%.

[0036] In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity for detecting the vaccination in the subject compared to a second sensitivity of detecting the vaccination in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, the sensitivity for detecting binding between the antibody and the two or more recombinant pathogen polypeptides compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the sensitivity of detecting infected versus vaccinated subjects.

[0037] In some embodiments, the signal moiety is complexed with the immunoglobulin binding agent. In some embodiments, the signal moiety comprises an enzyme, such as a horseradish peroxidase (HRP). In some embodiments, the signal moiety comprises a fluorescent molecule.

[0038] In some embodiments, the signal moiety and immunoglobulin binding agent may be components of an enzyme-linked immunosorbent assay (ELISA).

[0039] In some embodiments, a system for detecting an antibody produced in a subject in response to an infection from a pathogen further includes a therapeutic regimen for treatment against the infection from the pathogen or a vaccine for protection against the pathogen.Methods and workflow

[0040] The disclosure provides methods for detecting, in a sample obtained from a subject, an antibody response to a pathogen. In various embodiments, a method for detecting an antibody response to a pathogen may include: (a) capturing an antibody in the sample by contacting the sample with two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5), wherein the antibody binds to any one of the recombinant pathogen polypeptides; and (b) detecting binding between an antibody in the sample and the two or more recombinant polypeptides.

[0041] In some embodiments, the method comprises capturing an antibody in a sample derived from a subject by contacting the sample with the composition or the system described herein,Attorney Docket No.10620-163W01wherein the antibody binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5). In some embodiments, the method comprises contacting a sample derived from a subject with the composition or the system described herein; and detecting binding between an antibody in the sample and the two or more recombinant pathogen polypeptides disclosed herein.

[0042] In some embodiments, the antibody binding to the two or more recombinant pathogen polypeptides comprises an endogenous antibody produced by a subject in response to a pathogen or a component of the pathogen. In some embodiments, the antibody may be a canine antibody. In some embodiments, the antibody may be a human antibody.

[0043] In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. The two or more cytoplasmic domains of the recombinant pathogen polypeptides may, for example, comprise immunodominant amino acid sequences selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, hydrophilicity' prediction, or a combination thereof. In some embodiments, the two or more recombinant pathogen polypeptides may be attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides. In some embodiments, the two or more recombinant pathogen polypeptides are 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ I D NO: 15. In some embodiments, the two or more recombinant pathogen polypeptides are about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atAttorney Docket No.10620-163W01least 97%, at least 98%, or at least 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In some embodiments, the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15.

[0044] In some embodiments, detecting binding between an antibody in the sample and the two or more recombinant polypeptides includes: (a) contacting the sample with an immunoglobulin binding agent, wherein (i) the immunoglobin binding agent binds to an antibody binding to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5); and (ii) the immunoglobulin binding agent is complexed to a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding between the antibody and the two or more recombinant pathogen polypeptides.

[0045] In some embodiments, the immunoglobulin binding agent binds to an IgG antibody binding to the two or more recombinant pathogen polypeptides. The IgG antibody may, for example, be an IgGl, IgG2, IgG3, or IgG4 antibody.

[0046] In some embodiments, the signal moiety comprises an enzyme, such as a horseradish peroxidase (HRP). In some embodiments, the signal moiety comprises a fluorescent molecule. In some embodiments, the signal moiety and immunoglobulin binding agent may be components of an enzyme-linked immunosorbent assay (ELISA).

[0047] FIG. 1 is a flow diagram of an example of an assay workflow 100 for detecting, in a sample obtained from a subject, an antibody response to a pathogen. In one embodiment, assay workflow 100 may be used for the detection of an antibody produced by the subject in response to an infection from a pathogen. In one embodiment, assay workflow 100 may be used for detecting an antibody produced by a subject in response to vaccination against a pathogen of interest. Workflow 100 may include, but is not limited to, the following steps.

[0048] At a step 110, a sample is obtained from a subject for detection of an antibody produced in response to a pathogen of interest. The sample may, for example, be a blood sample or a component of the blood sample, such as a serum sample. In one embodiment, the pathogen of interest is a Bartonella species.Attorney Docket No.10620-163W01

[0049] At a step 115, the sample is contacted with two or more recombinant pathogen polypeptides selected to detect an antibody produced by the subject in response to the pathogen of interest. In some embodiments, the two or more recombinant pathogen polypeptides are selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5). Binding of an antibody in the sample to the two or more recombinant pathogen polypeptides produces an antibody / polypeptide complex.

[0050] At a step 120, the sample is contacted with an immunoglobulin binding agent functionalized with a signal moiety. In one embodiment, the signal moiety comprises horseradish peroxidase (HRP). Binding of the immunoglobulin binding agent to the antibody-polypeptide complex forms a binding agent / antibody / polypeptide complex.

[0051] At a step 125, the sample is contacted with a signal moiety substrate for detection of a binding agent / antibody / polypeptide complex. In some embodiments, the signal moiety is HRP and the detection substrate is a chromogenic tetramethylbenzidine (TMB) solution. Detection of a chromogenic signal generated by the signal moiety is indicative of binding among: an antibody; the two or more recombinant pathogen polypeptides; and the immunoglobulin binding agent.

[0052] At a step 130, a signal generated by the signal moiety is detected, wherein detection of a signal is indicative of binding among an antibody, the two or more recombinant pathogen polypeptides and the immunoglobulin binding agent.

[0053] In some embodiments of method 100, an antibody is produced by a subject in response to an infection from a pathogen. In this case, detecting binding between the antibody and the two or more recombinant pathogen polypeptides indicates an infection from a pathogen in the subject. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity for detecting the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to the second specificity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%. In some embodiments, detectingAttorney Docket No.10620-163W01binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity for detecting the infection in the subject compared to a second specificity for detecting the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity for detecting the infection in the subject compared to the second specificity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%.

[0054] In some embodiments, method 100 further comprises treating the subject infected with the pathogen.

[0055] In some embodiments of method 100, an antibody is produced by a subject in response to vaccination against a pathogen of interest (e.g., protecting the subject against the pathogen). In this case, detecting antibody binding to the two or more recombinant pathogen polypeptides indicates vaccination against a pathogen in the subject. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the vaccination in the subject compared to a second specificity for detecting the vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the vaccination in the subject compared to the second specificity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity for detecting the vaccination in the subject compared to a second sensitivity for detecting the vaccination in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity for detecting the vaccination in the subject compared to the second sensitivity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%.

[0056] In some embodiments of method 100, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the sensitivity of detecting infectedAttorney Docket No.10620-163W01versus vaccinated subjects. In some embodiments, detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity for detecting infected versus vaccinated subjects compared to the second sensitivity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 500%.Kit

[0057] The disclosure provides a kit for detecting an antibody response to a pathogen or a component of a pathogen of interest, the system comprising: (a) two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); (b) an immunoglobulin binding agent; (c) a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding among: an antibody; the two or more recombinant pathogen polypeptides; and the immunoglobulin binding agent; and (d) an instruction manual.

[0058] In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more immunodominant amino acid sequences. In some embodiments, the two or more recombinant pathogen polypeptides are attached to a solid support. In some embodiments, the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides. In some embodiments, the two or more recombinant pathogen polypeptides are 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant pathogen polypeptides are about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any two or more polypeptides selected from the group consisting of: SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15. In some embodiments, the signal moiety (c) is complexed with the immunoglobulin binding agent (b). In some embodiments, the signalAttorney Docket No.10620-163W01moiety comprises an enzyme, such as a horseradish peroxidase (HRP). In some embodiments, the signal moiety comprises a fluorescent molecule. In some embodiments, the kit further includes a therapeutic regimen for treating an infection from a pathogen or a vaccine for protection against the pathogen.

[0059] In some embodiments, a kit for detecting an antibody response to a pathogen comprises two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5) for detection of a Bartonella species.

[0060] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

[0061] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0062] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0063] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0064] Any systems, methods, software, and platforms described herein are modular.Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.

[0065] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (orAttorney Docket No.10620-163W01within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term “about” refers to ±10% of a stated number or value.

[0066] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0067] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[0068] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be constiued in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions,Attorney Docket No.10620-163W01configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.EXAMPLES

[0069] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way.Example 1: Development and Validation of Enzyme-Linked Immunosorbent Assays for the Serodiagnosis of Canine Bartonelloses

[0070] An immunoproteomic approach was used to identify immunodominant antigens that can be used for detection of anti-Bartonella antibodies in a subject sample. Specifically, we examined the diagnostic utility of five recombinant B. henselae antigenic targets: ATP-p, ATP synthase subunit beta; GroEL, a heat shock protein; LemA, a membrane protein; SucB, dihydrolipoaide succinyltranferase protein; and VirB5, putative component of type IV secretion system. The strains and plasmids used in this study for cloning, expression or purification of recombinant B. henselae proteins are shown in Table 1.

[0071] The sensitivity and specificity of the five B. henselae immunodominant recombinant proteins (rATP-P, rGroEL, rLem A, rSucB, and rVirB5) were evaluated in an ELISA format using serum samples from dogs. The dogs comprised two groups: Group I: 36 Bartonella spp. naturally infected dogs (PCR+ and all B. henselae IF A seroreactive) and Group II: 34 Bartonella spp. PCR negative and IFA negative dogs. Seventy archived sera from dogs previously tested at the North Carolina State University, College of Veterinary Medicine, Vector Borne Diseases Diagnostic Laboratory (NCSU-CVM-VBDDL) or the Intracellular Pathogens Research Laboratory, NCSU-CVM (NCSU-CVM-IPRL) were selected for comparative ELISA testing utilizing each of the purified recombinant B. henselae immunodominant proteins as previously described by Neupane, P. et al., Pathog. (Basel, Switzerland) 11, (2022), which is incorporated herein by reference in its entirety. Serum samples were categorized into two groups to assess assay sensitivity and specificity. All sera were stored frozen at -80 °C after being submitted to the NCSU-CVM-VBDDL for diagnostic testing between 2016 and 2020. In brief, Group IAttorney Docket No.10620-163W01consisted of 36 stored frozen serum samples from Bartonella spp. naturally infected dogs (Bartonella IFA-positive). A cutoff titer of >1:64 was used to define an IFA seroreactive titer. Group II consisted of 34 dogs for which diagnostic testing in the NCSU-CVM-VBDDL and NCSU-CVM-IRPL did not provide evidence of exposure to or infection with a Bartonella spp. These sera were used to partially assess the specificity of ELISA assays. These sera were IFA negative (titers <1:16) to the 3 Bartonella spp. (B. henselae San Antinio type 2, B. vinsonii subsp. berkhoffii genotype I, and B. koehlerae). All 34 Group II dogs were PCR negative after whole blood DNA extraction for Bartonella spp.Identification of B. henselae immunodominant proteins recognized by sera from experimentally and naturally infected dogs

[0072] In a previous study using Western blot (WB) analysis, we identified six B. henselae proteins (proteins of 13, 17, 29, 50, 56, and 150 kDa) that appeared to represent Bartonella relevant immunodominant antigens (see Neupane, P. et al., J, Clin. Microbiol, 58, (2020), which is incorporated herein by reference in its entirety). These B. henselae immunodominant proteins were identified using an in-house matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, ABSCIEX TOF / TOF 5800 mass spectrometer) as previously described in Kulkarni, M. M. et al., Retrovirology 14, 45 (2017), which is incorporated herein by reference in its entirety. M ALDI-MS / MS data were acquired using the AB Sciex 5800 TOF / TOF Mass Spectrometer (AB Sciex, Framingham, MA). Peptide mass fingerprint and peptide sequence data were resolved by the SPROT (UNIPROT) and NCBI databases using the Mascot search engine. Briefly, heat-denatured B. henselae SA2 whole cell proteins were separated by sodium dodecyl sul fate-polyacrylamide gel electrophoresis (SDS-PAGE) in Criterion precast gels, using 4 to 15% gradient polyacrylamide Tris-glycine precast midigels (Bio-Rad, Hercules, CA). The gels were fixed and silver stained according to established protocols.

[0073] FIG. 2 is a photograph of an example of an SDS-PAGE gel showing bands representing immunodominant proteins of 13, 17, 50, 56, and 150 KDa. Heat-denatured B. henselae SA2 whole cell proteins were separated by SDS-PAGE followed by silver staining of the gel. The majority of experimentally and naturally-infected dogs recognized Bartonella species proteins of molecular weights 13, 17, 50, 56, and 150 kDa (lanes 1 and 2), which suggests that these proteins appeared to be Bartonella relevant immunodominant proteins. Bands representing the immunodominant proteins were manually excised from the gel and in-gel digested with trypsinAttorney Docket No.10620-163W01(0.6 μg). Tryptic peptides were subjected to MALDI-MS on an ABSCIEX TOF / TOF® 5800 mass spectrometer for identification.

[0074] In addition, bands representing proteins of molecular weight 62 kDa and 50 kDa were also excised and subjected to MALDI-MS on an ABSCIEX TOF / TOF® 5800 mass spectrometer. In addition, bands representing proteins of molecular weight 62 kDa and 50 kDa were excised and were subjected to MALDI-MS on an ABSCIEX TOF / TOF® 5800 mass spectrometer. The proteins of molecular weights of 62 kDa and 50 kDa were included in this study. After intradermal inoculation of B. henselae, the 62 kDa was recognized by 30% of experimentally inoculated dogs. The 50 kDa protein was closest to the 52 kDa immunodominant protein that was recognized by dog sera in our Western blot study.

[0075] Based on peptide sequence and peptide fingerprint data, the B. henselae immunodominant proteins identified from each band are summarized in Table 2. All proteins listed in Table 2 achieved the minimum criteria for a single peptide with 99% confidence. The 56 kDa and 150 kDa bands were identified as B. henselae GroEL protein (NCBI Reference Sequence WP 034454894.1), a heat shock protein, and DNA-directed RNA polymerase subunit beta (WP_011180534.1), respectively. With the exception of 56 kDa and 150 kDa bands, more than two proteins were identified from each excised band (see Table 2). MS analysis of the 13 kDa band failed, probably due to a minimum protein content.

[0076] Among the B. henselae immunodominant proteins, each protein of a molecular weight of 17, 50 and 56 kDa yielded a sensitivity of >35% and specificity of >92% for canine Bartonelloses as determined by reactivity of these protein bands with sera from naturally infected (Bartonella PCR positive) compared to negative control dogs as previously described (see Neupane, P. et al., J. Clin. Microbiol. 58, (2020)). Of the several proteins identified from 17 kDa and 50 kDa bands, we selected B. henselae LemA and rATP-P for further study, based on reactivity patterns of these immunodominant patterns as described in Vigil, A. et al, PLoS One 5, el 1447 (2010); Saisongkorh, W. et al., FEMS Microbiol. Lett. 310, 158-67 (2010); and Boonjakuakul, J. K. et al,, Infect. Immun. 75, 2548-61 (2007), which are incorporated herein by reference in their entirety. Bartonella henselae GroEL was the only identified protein from the 56 kDa gel band that was evaluated in this study. Previous studies reported GroEL as a B. henselae immunodominant protein that may be a suitable diagnostic marker for Bartonelloses (see Saisongkorh, W. et al (2010); and McCool, T. L. et al., Diagn. Microbiol. Infect. Dis. 60, 17-Attorney Docket No.10620-163W0123 (2008), which is incorporated herein by reference in its entirety). Two additional B. henselae immunodominant proteins (SucB and VirB5) were selected through literature mining (see, Saisongkorh, W. et al (2010); and Okaro, U. et al., Clin. Microbiol. Rev. 30, 709-746 (2017) and Anderson, B. et al., J, Clin. Microbiol. 33, 2358-65 (1995), which are incorporated herein by reference in their entirety). These prior studies also suggested that SucB and VirB5 may be potential diagnostic candidates for diagnosis of Bartonelloses. Based on results from WB analysis and literature mining, we elected to evaluate the diagnostic utility of five B. henselae recombinant proteins (rATP-P, rGroEL, rLemA, rSucB, and rVirB5).Evaluation of B. henselae peptides and immunodominant proteins for development of diagnostic ELISAs

[0077] To assess the potential diagnostic utility of ATP-P, GroEL, and LemA proteins identified from our Western blot study, the Immune-Epitope-Database and Analysis-Resource (IEDB) antibody epitope prediction tool (BepiPred 2.0) with default parameter settings was applied to the B. henselae ATP-P, GroEL, and LemA protein sequences as describe in Sanchez-Trincado, J. L., et al., J. Immunol. Res. 2017, 2680160 (2017); and Jespersen, M. C., et al., Nucleic Acids Res. 45, W24-W29 (2017), which are incorporated herein by reference in their entirety. Based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, and hydrophilicity prediction (http: / / tools.iedb.org / bcell / ), B. henselae F0F1 ATP synthase subunit beta (NCBI Reference Sequence WP_082251662.1; ranging from 109 to 265 amino acids), B. henselae GroEL (NCBI Reference Sequence: WP_034454894.1; ranging from 193 to 370 amino acids) and B. henselae LemA protein (NCBI Reference Sequence: WP 011180688.1; ranging from 79 to 214 amino acids), all comprising high scores for predicted B-cell epitopes, were selected for further analysis. In addition, the full-length recombinant SucB and VirB5 proteins were selected for further evaluation. The selected regions of ATP-P, GroEL, and LemA, and full-length recombinant SucB and VirB5 are designated rATP-P, rGroEL, rLemA, rSucB, and rVirB5.

[0078] FIG. 3 is a panel of plots (A), (B), and (C) showing predicted B-cell epitopes of Bartonella henselae F0F1 ATP synthase subunit beta, B. henselae GroEL and B. henselae LemA protein, respectively, obtained using the IEDB antibody epitope prediction tool. Panel (A) shows predicted B-cell epitopes of Bartonella henselae F0F1 ATP synthase subunit beta (NCBI Reference Sequence WP 082251662.1; ranging from 109 to 265 amino acids). Panel (B) showsAttorney Docket No.10620-163W01predicted B-cell epitopes of B. henselae GroEL (NCBI Reference Sequence: WP 034454894.1; ranging from 193 to 370 amino acids). Panel (C) shows predicted B-cell epitopes of B. henselae LemA protein (NCBI Reference Sequence: WP 011180688.1; ranging from 79 to 214 amino acids). The immunodominant peptide regions of ATP-p, GroEL and LemA are represented by the boxes in FIG. 2. Based on immunoproteomic analysis, these peptide regions of B. henselae ATP-P, GroEL, and LemA were selected for cloning, expression, purification, and evaluation for use in a diagnostic assay. A summary of the peptide regions selected for further analysis is shown in Table 3.Amplification of B. henselae A TP synthase subunit beta atpD a heat shock protein (groEL), a membrane protein (lemA), dihydrolipoaide succinyltranferase protein (sucB), and putative component of type IV secretion system (virB5)

[0079] The full-length sucB and virB5 and selected regions of atpD, groEL, and lemA genes were PCR-amplified, cloned and expressed using an Escherichia coli expression system.Genomic DNA of B. henselae SA2 was extracted from blood agar plate grown B. henselaeusing DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) following the manufacture’s protocol. The primer sets used for conventional PCR amplification of the sucB, virB5, atpD, groEL, and lemA genes using B. henselae SA2 are shown in Table 4. Conventional PCR was performed in a 25 pL final volume reaction containing 12.5 μL of Q5 High-Fidelity 2X Master mix (New England Biolabs, USA, cat. No. M0492S), 0.2 μL of 100 μM of each forward and reverse primer (IDT-DNA Technology), 7.3 μL of molecular-grade water, and 5 μL of DNA from each sample tested. Genomic DNA from B. henselae Houston- 1 was used as a positive control. Five μL of Ultra-Pure, molecular grade water (Genesee Scientific, San Diego, CA, USA) and 5 μL of DNA extracted from Escherichia coli were used as negative controls. Conventional PCR was performed in an Eppendorf Mastercycler EP gradient under the following conditions: a single hot-start cycle at 95 °C for 2 min followed by 30 cycles of denaturing at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. Amplification was completed by an additional cycle at 72 °C for 2 min. Prior to the ligation reaction for cloning, PCR products were purified by gel extraction using PureLink quick gel extraction and PCR purification combo kit (Invitrogen, Carlsbad, CA).Attorney Docket No.10620-163W01Cloning, expression, and purification of recombinant ATP-β, GroEL, LemA, SucB, and VirB5

[0080] The PCR amplified atpD, groEL, lemA, sucB, and virB5 genes were inserted into the Champion™ pET200 Directional TOPO® Expression kit (Invitrogen, Carlsbad, CA) and transformed into E. coli chemically competent TOPIO cells. The recombinant plasmids were then purified and insert sequences were confirmed by Sanger sequencing (Genewiz, Research Triangle Park, NC). Recombinant plasmids constructs were then transformed into BL21 Star™ (DE3) (Invitrogen, Carlsbad, CA) and induced at 1 mM IPTG for protein purification.Expression of recombinant products were verified by resolution of total crude protein by SDS-PAGE followed by Western blot analysis using Pierce™ 6X-His Epitope-Tag mouse monoclonal antibody (Thermo Scientific, Rockford, IL) and anti-mouse IgG secondary antibody (Rockland, Gilbertsville, PA). IPTG-induced cell pellets were subjected to BugBuster Master Mix (EMD Millipore Corp., Billerica, MA) treatment and soluble fractions containing His-tagged proteins were purified using HisPur™ Cobalt spin columns (Thermo Scientific, Rockford, IL) according to manufacturer’s instructions. Induced recombinant proteins were purified by column¬ chromatography using HisPur™ Cobalt spin columns according to manufacturer’s instructions (Thermo Scientific, Rockford, IL). Fractionated proteins were visualized by staining the gel overnight with Bio-safe™ Coomassie brilliant blue (Bio-Rad, Hercules, CA). Purified recombinant proteins were verified by WB using Pierce™ 6X-His Epitope-Tag mouse monoclonal antibody (Thermo Scientific, Rockford, IL) and anti-mouse IgG secondary antibody (Rockland, Gilbertsville, PA), Proteins were dialyzed into phosphate buffered saline (PBS; overnight) using Slide-A-Lyzer™ Dialysis Cassettes, (2-10 kDa MWCO cutoff; Thermo Scientific, Rockford, IL).

[0081] To evaluate the sensitivity and specificity of purified recombinant ATP-P, GroEL, LemA, SucB, and VirB5, an ELISA protocol was performed using sera from Group I (n=36), and II (control group; n=34) dogs. Each protein was immobilized in duplicate in ELISA plate wells (500 ng / well) using carbonate buffer as described previously by Neupane, P et al., Pathog. (Basel, Switzerland) 11, (2022), which is incorporated herein by reference in its entirety. Briefly, ELISA plates were coated with 100 μL of 10 μg / mL recombinant purified proteins in carbonate buffer, pH 9.6, overnight at 4°C. After each incubation time, plates were washed four times using IX PBS-T washing buffer (IX PBS with 0.05% Tween 20). After washing, plates were blocked with 1.5% nonfat milk powder in IX PBS (blocking solution) for 2 h at room temperature (RT).Attorney Docket No.10620-163W01After washing, sera from dogs in 1:100 blocking solution added and incubated for 1 hour at RT. After washing four times with I X PBST, plates were incubated with 100 pL of HRP-conjugated goat anti-dog IgG (1:2,000 dilution; Invitrogen, Carlsbad, CA) (1:5,000; Abeam, Cambridge, MA) in blocking solution. The plates were developed with addition of 50 pL of 1-Step™ Ultra TMB ELISA substrate solution (Invitrogen, Carlsbad, CA) for 15 min followed by addition of 2M H2SO4to stop the reaction. Absorbance values at 450 nm were measured using a Tecan plate reader. Sera from dogs naturally infected with B. henselae (B. henselae IF A titer >1: 512) and Bartonella PCR-negative and IFA-negative dog sera were used as positive and negative controls, respectively. Plate wells coated with only coating buffer (without protein) were used as blanks to determine the subtracted background noise. Based on results from the negative controls, a baseline was established for scoring individual samples as positive or negative for Bartonella exposure. The average absorbance value was calculated for each set of duplicate samples.

[0082] Receiver operating characteristic (ROC) analysis was performed with 95% Cis to determine sensitivity, specificity, ELISA cutoff values, and to distinguish reactivity between positive and control groups, as previously described by Youden, W. J., Cancer 3, 32-35 (1950); and Schisterman, E. F., et al., Stat. Med. 27, 297-315 (2008), which are incorporated herein by reference in their entirety. Differences in IgG reactivity to target proteins between infected and control groups were analyzed using Mann-Whitney U test. Optimal density (OD) cutoff values were determined to maximize Youden index as previously described. Scatterplots of ELISA OD values were generated to determine differences in ELISA seroreactivity between Bartone Ila infected and control groups. To compare the serological results obtained from ELISA for all groups, overall percent agreements between Bartonella spp. IF A and ELISA were calculated as described previously in Gardner, LA., et al., Prev. Vet. Med. 45, 107-22 (2000), which is incorporated herein by reference in its entirety. Scatterplots and ROC curves were generated in Windows 10 operating system with the help of. Anaconda Navigator version 1.9.12. The scatter plots were generated using Python 3. 6.13 in Jupyterlab 3.2.1 and the ROC curves were analyzed using R version 3.6.1 in R studio 1.1.456 (accessed on October 15, 2024). p values of less than 0.05 were considered statistically significant.

[0083] Sanger sequencing confirmed in-frame insertion of coding sequences of atp-, groEL, lemA, sucB, and virB5, in pET200D / TOPO expression system as determined by ampliconAttorney Docket No.10620-163W01sequence of the plasmid isolated from the respective recombinant E. coll BL21 [DE3] clones. Purified proteins were confirmed by Coomassie stained SDS-PAGE and Western blot analysis.

[0084] FIG. 4 is a panel of photographs showing an example of Coomassie stained SDS-PAGE and Western blot analysis of purified recombinant proteins rATP-P, rGroEL, rLemA, rSucB, and rVirB5. In this example, Western blot analysis of purified proteins was performed using mouse anti-His antibody (Thermo Scientific, Rockford, IL) and alkaline-phosphatase conjugated Goat anti-mouse IgG (Thermo Scientific, Rockford, IL). Predicted molecular mass of recombinant proteins were determined by coding sequence of the specified gene insert plus fused in frame with pET200D / TOPO expression system fusion tag (~3 kDa) as indicated by red arrows. The green arrows indicate recombinant proteins in multimeric forms. Coomassie= Coomassie stained SDS-PAGE; Anti-His= Western blot analysis; and L= protein ladder.

[0085] The sensitivity and specificity of each of the five Bartonella recombinant proteins were assessed by ELISA testing using sera from Group I (n = 36) naturally infected dogs and Group II (n = 34) control dogs. ELISA OD cutoff values were determined to maximize sensitivity and specificity for each recombinant protein at maximal Youden index.

[0086] FIG. 5 is a panel of scatter plots (A), (B), (C), and (D) of the ELISA seroreactivity of recombinant proteins (rATP-P, rGroEL, rLemA, rSucB, and rVirB5, respectively) among Bartonella-infected and control dogs. Panel (A) shows the ELISA seroreactivity for recombinant rATP-P; panel (B) shows the ELISA seroreactivity for recombinant rGroEL; panel (C) shows the ELISA seroreactivity of recombinant rLemA; panel (D) shows the ELISA seroreactivity of recombinant rSucB; and panel (E) shows the ELISA seroreactivity of recombinant rVirB5. Respective p values (dotted line) between sample groups are given. Optical density cutoff values as determined at maximum Youden Index are represented by the solid black line.

[0087] FIG. 6 is a panel of plots (A), (B), (C), (D), and (E) showing receiver operating characteristic ( ROC ) curves with 95% Cis for ELISA seroreactivity of Bartonella recombinant proteins (rATP-P, rGroEL, rLemA, rSucB, and rVirB5, respectively) for dogs. Panel (A) shows ROC curves for recombinant rATP-p; panel (B) shows ROC curves for recombinant rGroEL; panel (C) shows ROC curves for recombinant rLemA; panel (D) shows ROC curves for recombinant rSucB; and panel (E) shows ROC curves for recombinant rVirB5. Cutoff values were determined to maximize Youden index. For each recombinant protein, false positive andAttorney Docket No.10620-163W01true positive are shown in parentheses, respectively, at the intersection of dotted line. AUC= Area under curve.

[0088] Referring now to FIG. 5 and FIG. 6, of the Bartonella recombinant proteins, rGroEL resulted in an 83% sensitivity and 94% specificity at a cutoff OD value of 0.439. In addition, rGroEL yielded the highest AUC score of 0.93 (95% CI 0.87-0.99) (see FIG. 6). The sensitivity and specificity of recombinant rATP-P was 69% and 94% at a cutoff value of 0.565. Only two Group II control dogs (0.058%) were reactive to rATP-P at the cutoff value of 0.565. Two Group II dogs were positive for rGroEL at the cutoff 0.439, one of which was also positive for rATP-p. These three dogs were IFA seroreactive to Rickettsia rickettsii. Of the three Group II control dogs that were reactive to rATP-P or rGroEL at the given cutoff values for respective recombinant proteins, one dog was ELISA positive for both rATP-P and rGroEL proteins.

[0089] The true positive and false positive rates for rVirB5 were 72% and 29%, respectively, and AUC score for rVirB5 was 0.76 (95% CI, 0.647-0.872). Although sensitivity of both rLemA and rSucB was >70%, more than 30% of Group II control dogs were reactive to rLemA and rSucB above their respective cutoff values (see FIG. 5). Significantly more recombinant proteins that bound IgG were detected in sera from Group I infected dogs as compared with control dogs for ail the recombinant proteins (see FIG. 5, Mann-Whitney U test, p< 0.05). rATP-P and rGroEL based ELISA had AUC scores greater than 0.8 whereas AUC scores for all other recombinant proteins were less than 0.75. Based on all the diagnostic parameters tested (sensitivity, specificity, and AUC score), rATP-p and rGroEL represented the optimal candidates for the serodiagnosis of Bartonella infection in dogs.

[0090] Since rATP-p and rGroEL were the most reactive proteins, we used a combination of rATP-β and rGroEL to test 34 Group I (inadequate volume for 2 / 36 Group I sera) and 34 Group II control dogs. FIG. 7 is a scatter plot (A) and ROC curves (B) for ELISA seroreactivity of Bartonella recombinant proteins rATP-P and rGroEL used in combination. Cutoff values were determined to maximize Youden index. For each recombinant protein, false positive and true positive are shown in parentheses, respectively, at the intersection of dotted line. AUC= Area under curve.

[0091] By combining these two proteins, sensitivity was 88% and specificity 92% at an OD cutoff value of 0.505 at maximum Youden index. At an OD cutoff of 1.195 (trade-off between sensitivity and specificity), sensitivity was 74% and specificity was 100%. Of the three rATP-PAttorney Docket No.10620-163W01plus rGroEL ELISA positive Group II dogs, one was reactive to both rATP-P and rGroEL, one was only reactive to rGroEL and the remaining dog was not reactive to either rATP-P or rGroEL. A ROC curve analysis for the rATP-P plus rGroEL yielded AUG score of 0.899 (95% CI 0.809-0,989), There was significant difference (p::::2e,08) in the rATP-P plus rGroEL reactivity (p:::0) between Group I infected and Group II control dogs.Comparison of Bartonella spp. IFA results and ELISA reactivity for dogs

[0092] Since IFA is the most frequently used “gold standard” method for screening anti-Bartonella antibodies in serum samples from dogs, we compared diagnostic agreements between Bartonella spp. IFA and ELISA results for each immunodominant protein. The comparison between Bartonella IFA and rATP- plus rGroEL ELISA test results for arhx-Bartonella antibodies in serum samples from dogs is summarized in Table 5. Group I dogs (n=36) were naturally infected with Bartonella spp. All Group I dogs were B. henselae IFA seroreactive (IFA titer >1 64). Group II dogs consisted of 34 Bartonella spp, IFA negative and PCR negative control dogs. For the rATP-P plus rGroEL ELISA, 34 Group I dogs were used due to inadequate serum volumes from two dogs. Based on comparison of IFA and ELISA results in dogs, there was substantial agreement (overall 90% agreement; kappa value =0.794) between Bartonella IFA and rATP- plus rGroEL ELISA. Thirty (83%) of B. henselae IFA positive Group I naturally infected dogs were positive by the rATP-p plus rGroEL ELISA, while 32 of 34 IFA negative Group II dogs were also negative by the rATP-P plus rGroEL ELISA. The overall proportion agreement between Bartonella IFA and rATP-p or rGroEL was substantial (kappa value =0.63-0.8). Based on calculation of overall agreement between IFA and ELISA for dogs, the overall proportion agreement was 88% for Bartonella IFA and rATP-p ELISA and 81% and for Bartonella IFA and rGroEL ELISA.

[0093] While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually and separately indicated to be incorporated by reference for all purposes.Attorney Docket No.10620-163W01TablesTable 1. Bacterial strains and plasmids used for cloning, expression or purification of recombinant B. henselae proteins.Source or Strain or Plasmids DescriptionReference Bartonella henselae San *Feline originAntonio 2Champion™ pET200Directional TOPO™ Invitrogen Expression KitF- mcrA E(mrr-hsdRMS-mcrBC)One Shot™ TOP 10O80 / acZAM15 A / acX74 recAA araD139chemically competent Invitrogen A( araleu)'1691 galU galK rpsLEscherichia coli(StrR) end Al mipGBL21 Star™(DE3) oneF-ompF hsdSB (rB-, mB-) galdcmrne 131Shot™ Chemically Invitrogen (DE3)competent Escherichia colipET2007D-TOPO Vector Invitrogen pUC57 Novagen pET46Ek-LIC vector Novagen endAl hsd. R17 ( rr; 12 mKi2h) sitpE44 thi-lE. coli NovaBLUE cells recAl gyrA96 relAl lac Novagen F'|>roAdB+lacRZMdl5:-. TnlO] (TetR)BL21 (DE3) F - ompT hsdSulpB IHB ) gal dem (DE3) Novagen*Described in Neupane, P. et a., J. Vet. Intern. Med. 32, 1958=1964 (2018), which is incorporated herein by reference in its entirety.Table 2. Identification of Bartonella henselae immunodominant proteins by the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). All proteins listed achieved the minimum criteria of one peptide with 99% confidence. With the exception ofAttorney Docket No.10620-163W0156 and 150 kDa proteins, more than two proteins were identified from each gel band. MALDI-MS / MS data were acquired using the AB Sciex 5800 TOF / TOF Mass Spectrometer (AB Sciex, Framingham, MA).WB Band (kDa) Protein Identification by MALDI-TOF MS13 N / ALemA Family protein (WP 011180688.1), Invasion associated protein lalB (WP_034454864), Peptidylpropyl isomerase (Bh_011180874.1), 50S 17ribosomal protein L6 (WP 082250521), Transcription factor GreA (WP_034447961),ATP Synthase subunit alpha (WP_082251272.1), ATP Synthase subunit 50beta (WP_082251662.1), GroEL (WP 034454894.1)GroEL (WP_034454894.1), ATP Synthase subunit beta 52(WP 082251662.1), dihydrolipoyl dehydrogenase (Bh__WP__082252214.1) 56 GroEL (WP 034454894.1)Succinate dehydrogenase flavoprotein subunit (SdhA; WP_034454472.1) 62Peptidylprolyl isomerase (WP_082252249.1)150 DN A-directed RN A polymerase subunit beta (WP_011180534.1 )Table 3. Regions Bartonella henselae (ATP-P), a heat shock protein (GroEL), a membrane protein (LemA), dihydrolipoaide succinyltranferase protein (SucB), and putative component of type IV secretion system (VirB5) that were selected for cloning, purification, and expression in this study. aa= amino acids.Proteins NCBI Reference Selected Designated Moi. Reference Sequence (Length; mol Region for Recombinant Mass mass) Cloning Protein Name (kDa) of (location) SelectedRegionAttorney Docket No.10620-163W01ATP-p WP_082251662.1 (531 157 (109-265 rATP-P 16.52 This aa; 56.59 kDa) aa) study GroEL WP_034454894.1 (547 178 (193-370 rGroEL 19.59 This aa; 57,65 kDa) aa) study LemA WP 011180688.1 (214 136 (79-214 rLemA 14.1 This aa; 24.50 kDa) aa) study SucB AAR21287.1 (406 aa; 406 (1-406 rSucB 43.65 This 43.46 kDa) aa) study VirB5 WP 038488324.1 (148 147 (1-147 rVirB5 16.9 This aa; 16,9 kDa) aa) studyTable 4. PCR primers used for cloning, expression, and purification of the Bartonella henselae San Antonio 2 atp-P, groEL, lemA, sucB, and virB5 genes using ChampionIMpET200 Directional TOPO® Expression kit. The four nucleotides in bold represent nucleotides that were added at the 5’ end of forward primer to enable directional cloning in the pET200 / D-TOPO vector.Recombinant Selected Region of Proteins Primers used for PCR amplification and Cloning proteins for Cloning and Purification (Sequence 5’ — » 3’)(NCBI Reference Sequence;selected amino acids (aa))rATP-P WP_082251662.1; 109 to BhATP-P-325F256 aa CACCGCCATGGATACTACCGATGGTCTTG BhATP-P-795R TTATCCTTCTGTTGAACCATTGTTGTCrGroEL WP 034454894.1; 193 to BhGroEL-577F370 aa CACC Al GCAGTTTGAl CGTGGA TATCTT TC BhGroEL-lllORTTAAGCAAGTCTTTCTTGCAATTTTTCAttorney Docket No.10620-163W01rLemA WP_011180688.1; 79 to BhLemA-235F214 aa CACCGCTACCCATGAACAAGCTGTTTTTAC BhLemA-645R TTAATTAAAATTAACCTTCGGTGTTTGrSucB AAR21287.1; 1 to 406 aa BhSucB-lF CACCATGACTACTGAAATCCGTGTTCCBhSucB-1221R TTACAAGTCAAGAACCAGGCGrVirB5 WP 038488324.1; 1 to 147 BhVirB5-lF33 CACCATGAAAAAATATAGCTTAGTCAC BhVirB5-447RCTAAAGTCGGACATCAGATTTTCCTable 5. Comparison of Bartonella IF A and ELISA results for dogs. Group I dogs (n=36) were naturally infected with Bartonella spp. All Group I dogs were B. henselae IF A seroreactive (IFA titer >1:64). Group II dogs consisted of 34 Bartonella spp. IFA negative and PCR negative control dogs. For the rATP-P plus rGroEL ELIS A, 34 Group I dogs were used due to inadequate serum volumes from two dogs.Dog Groups (I and II)IFA Result ELISA ResultrATP-BPositive NegativePositive (n=36) 25 11Negative (n=34) 2 32rGroELPositive NegativePositive (n=36) 30 6Negative (n=34) 2 32rATP-B plus rGroELPositive NegativeAttorney Docket No.10620-163W01Positive (n=34) 30 4Negative (n=34) 3 31rLemAPositive NegativePositive (n=36) 33 3Negative (n=34) 15 19rSucBPositive NegativePositive (n=36) 26 10Negative (n=34) 11 23rVirB5Positive NegativePositive (n=36) 26 10Negative (n=34) 10 24Pathogen polypeptide sequencesSEQ Amino acid sequence Bartonella NCBI ID henselae Reference NO proteins sequence SEQ MVKAVTSSKETAKVEKKKSAPRSGWKAVSKSQAG ATP-P WP 01118 ID VKDSSSPVHKSSKKAPLAEAAVGVIKQVIGAVVDVQ 1298.1 NO: FEGPLPNILNA. LETDNLGNRLVI., EVAQHLGENTVRTI1 AMDTTDGLVRGQKVFDTGTQISVPVGEATLGRIMN VIGEPVDNVGPIATSKTRSIHQEAPEYVEQSTASEILV TGIKVVDLLAPYSKGGKVGLFGGAGVGKTVLIMELI NNIAKAHGGYSVFAGVGERTREGNDLYYEMIESRV NVNPKDNNGSTEGSKC / VLVYGQMNEPPGARARVA LSGLTIAESFRDEGQDVLFFVDNIFRFTQ AGAEVS AL LGRIPSAVGYQPTLATDMGALQERITSTRTGSITSVQ AIYVPADDLTDPAPATSFAIILDATTVLSRSIAEKGIY PAVDPLDSFSRMLDPLIVGEEHYTVACQVQTILQRY RSLQDIIAILGMDELSEDDKLLVGRARKIERFLSQPFH VAEAFTGSPGKLVPLEETIKGFKGLCAGEYDNLPEAAFYMVGSIDEAVEKGKRLIAEVSSAttorney Docket No.10620-163W01SEQ Amino acid sequence Bartonella NCBI ID henselae Reference NO proteins sequence SEQ MAAKEVKFGREARERLLRGVDILANAVKVTLGPKG GroEL WPJH118 ID RNVVIDKSFGAPRITKDGVSVAKEIELEDKFENMGA 1151.1 NO: QMLREVASKTNDIAGDGTTTATVLGQAIVQEGVKA2 VAAGMNPMDLKRGIDAAVDEVVANLFKKAKKIQTSAEI AQVGTI S ANG AAEIGKMIA DAMEK VGNEG VIT V EEAKTAETELEVVEGMQFDRGYLSPYFVTNAEKMV ADLDDPYILIHEKKLSNLQSLLPVLEAVVQSGKPLLII AED VEGEA L ATLVVNK LRGGLKIA AVKA PGFGDRR KAMLEDIAILTSGQVISEDVGIKLENVTLDMLGRAK KVNISKENTTIIDGAGQKSEINARVNQIKVQIEETTSD YDREKLQERLAKLAGGVAVIRVGGATEVEVKEKKD RVDDALNATRAAVEEGIVAGGGTALLRAANALTVK GSNPDQEAGINIVRRALQAPARQIATNAGEEAAIIVG KVLENNADTFGYNTATGEFGDLIALGIVDPVKVVRS ALQNA / ASIASLLITTEAMVAEVPKKDTPVPPMPGGGMGGMGGMDF SEQ MLNVRNASIIFTYPMLKMLRRFSIPILLAFLIPFLSGC LemA WP-01118 ID GFNTIPTNEEKAHAAWSEX^LNQYQRRADLIPNLVET 0688.1 NO: VKAYATHEQAVFTNVVEARAKATQININADMLNNP3 EIMQQYLNDQANLSSALSRLMAVVENYPDLKANQN FLALQSQLEGTENRISVARRDYIETVRIYNTALKTMP TMLWAKLWFRDAKPMPTFTIDDNSQQTPKV’NFNSEQ MTTEIRVPTLGESVTEATVGKWFKKLGEAVAVDEPL SucB WP_01118 ID IELETDKVTVEVPSPVAGKLSEIIAKEGDTVEVKALL 1413.1 NO: GLVEAGAAGISQSFSPSATPIPEVPSELKQSSSSGAMQ4 KDTMPPSPSAAKLMAENNIAKSNISGSGKRGQILKE DVLGVLEQEVKAPSVSAASSSASLVQEKHEERVRM TKLRQTIARRLKDAQNTAAMLTTFNEVDMSAVMDL RKRYKDLFEKKHGVKLGFMGFFTKAVCHALKELPA VNAEIDGTDIVYKNYVNVGIAVGTDKGLVVPVVRH ADQMSLAEIEKEIGRLGRLARDGKLAVSDMQGGTF TITNGGVYGSLMSTPILNAPQSGILGMHAIKERAMV VGGQIVIRPMMYLALSYDHRIVDGQEAVTFLVRVKE SLEDPEJRLVLDL SEQ MKKYSLV1TLSLFCISH / AKAQTATLTDEYYKKALEN VirB5 WP-01118 ID TQKLDVAKSQTAESIYESATQTANKIKDINNQLANL 1130.1 NO: KADTKTKPEQLQALQIELTLLQAQLQADTLKIQSLA5 MIQAKDTKTKEELREEQTQKK. HEDLQKQLKEKLEKSDVRL SEQ AMDTTDGLVRGQKVFDTGTQISVPVGEATLGRIMN rATP-P WP 01118 ID VIGEPVDNVGPIATSKTRSIHQEAPEYVEQSTASEILV 1298.1; 109 NO: TGIKWDLLAPYSKGGKVGLFGGAGVGKTVUMELI to 265 aa 6Attorney Docket No.10620-163W01SEQ Amino acid sequence Bartonella NCBI ID henselae Reference NO proteins sequence NNIAKAHGGYSVFAGVGERTREGNDLYYEMIESRV NVNPKDNNGSTEGSEQ MQFDRGYLSPYFVTNAEKMVADLDDPYILIHEKKLS rGroEL WP-01118 ID NLQSLLPVT.. EAVVQSGKPLLIIAEDVEGEALATLWN 1151.1; 193 NO: KLRGGLKIAAVKAPGFGDRRKAMLEDIAILTSGQVIS to 370 aa 7 EDVG1KLENVTLDMLGR / AKKVNISKENTTIIDGAGQ KSEINARVNQIKVQIEETTSDYDREKLQERLA SEQ ATHEQAVFTNVVEARAKATQININADMLNNPEIMQ rLemA WP-01118 ID QYLNDQANLSSALSRLMAVVENYPDLKANQNFLAL 0688.1; 79 NO: QSQLEGTENRISVARRDYIETVRIYNTALKTMPTML to 214 aa 8 WAKLWFRDAKPMPTFTIDDNSQQTPKVNFNSEQ AAGISQSFSPSATPIPEVPSELKQSSSSGAMQKDTMPP rSucB WP 01118 ID SPSAAKLMAENNIAKSNISGSGKRGQILKEDVLGVL 1413.1; 80 NO: EQEVK AP S V S A AS S S ASLVQEKI IEERVRMTKLRQT to 188 aa 9SEQ VGIAVGTDKGLVVPVVRHADQMSLAEIEKEIGRLGR rSucB WP 01118 ID LARDGKLAVSDMQGGTFTITNGGVYGSLMSTPILNA 1413.1; 270 NO: PQSGILGMHAIKERAMVVGGQIVIRPMMYLALSYD to 394 aa 10 HRIVDGQEAVTFLVRVKESEQ AAGISQSFSPSATPIPEVPSELKQSSSSGAMQKDTMPP rSucB WP 01118 ID SPSAAKLMAENNIAKSNISGSGKRGQILKEDVLGVL 1413.1; (80 NO: EQEVK AP S V S A AS S S ASLVQEKI IEERVRMTKLRQT V to 188) 11 GIAVGTDKGLVVPVVRHADQMSLAEIEKEIGRLGRL (270 to ARDGKLAVSDMQGGTFTFFNGGVYGSLMSTP1LNAP 394) aa QSGILGMHAIKERAMVVGGQIVIRPMMYLALSYDH RIVDGQEAVTFLVRVKE SEQ AQTATLTDEYYKKALENTQKLDVAKSQTAESIYESA rVirB5 WP 01118 ID TQTANKIKD 1130.1; 20 NO: to 64 aa 12SEQ ANLKADTKTKPEQLQA rVirB5 WP 01118 ID 1130.1; 70 NO: to 85 aa 13SEQ LQADTLKIQSLAMIQAKDTKTKEELREEQTQKKHED rVirB5 WP 01118 ID LQKQLKEKLEKSD 1130.1; 97 NO: to 145 aa14Attorney Docket No.10620-163W01SEQ Amino acid sequence Bartonella NCBI ID henselae Reference NO proteins sequence SEQ AQTATLTDEYYKKALENTQKLDVAKSQTAESIYESA rVirB5 WP_01118 ID TQTANKIKDANLKADTKTKPEQLQALQADTLKIQSL 1130.1; (20 NO: AMIQAKDTKTKEELREEQTQKKHEDLQKQLKEKLE to 64) + (70 15 KSD to 85) + (97to 145) aaLIST OF EMBODIMENTS

[0001] The following list of embodiments of the invention are to be considered as disclosing various features of the invention, which features can be considered to be specific to the particular embodiment under which they are discussed, or which are combinable with the various other features as listed in other embodiments. Thus, simply because a feature is discussed under one particular embodiment does not necessarily limit the use of that feature to that embodiment.

[0002] Embodiment 1. A system comprising: two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-p (rATP-P), a recombinant GroEL (rGroEL), a recombinant Lem A (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); and an immunoglobulin binding agent, wherein the immunoglobin binding agent binds to an antibody, wherein the antibody binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5).

[0003] Embodiment 2. The system of embodiment 1, wherein the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains.

[0004] Embodiment 3. The system of embodiment 1 or 2, wherein the two or more recombinant pathogen polypeptides comprise i munodominant amino acid sequences.

[0005] Embodiment 4. The system of embodiment 3, wherein the two or more immunodominant amino acid sequences are selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, hydrophilicity prediction, or a combination thereof.

[0006] Embodiment 5. The system of any one of embodiments 1-4, wherein the two or more recombinant pathogen polypeptides are attached to a solid support.Attorney Docket No.10620-163W01

[0007] Embodiment 6. The system of any one of embodiments 1-5, wherein the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides.

[0008] Embodiment 7. The system of embodiment 1, wherein the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15.

[0009] Embodiment 8. The system of embodiment 7, wherein the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15.

[0010] Embodiment 9. The system of any one of embodiments 1-8, wherein the immunoglobulin binding agent binds to a IgG.

[0011] Embodiment 10. The system of embodiment 9, wherein the IgG comprises IgGl, IgG2, IgG3, or IgG4.

[0012] Embodiment 11. The system of any one of embodiments 1-10, further comprising a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding between the antibody and the two or more recombinant pathogen polypeptides.

[0013] Embodiment 12. The system of embodiment 11, wherein the signal moiety is complexed with the immunoglobulin binding agent.

[0014] Embodiment 13, The system of embodiment 11, wherein the signal moiety comprises an enzyme.

[0015] Embodiment 14. The system of embodiment 13, wherein the enzyme comprises a horseradish peroxidase (HRP).

[0016] Embodiment 15, The system of embodiment 11, wherein the signal moiety comprises a fluorescent molecule.

[0017] Embodiment 16. The system of any one of embodiments 1-15, wherein the antibody comprises an endogenous antibody.

[0018] Embodiment 17. The system of any one of embodiments 1-16, wherein the antibody binds to two or more of the recombinant pathogen polypeptides.

[0019] Embodiment 18. The system of any one of embodiments 1-17, wherein the antibody is produced in response to an infection from a pathogen.

[0020] Embodiment 19. The system of any one of embodiments 1-17, wherein the antibody is produced in response to vaccination against a pathogen.Attorney Docket No.10620-163W01

[0021] Embodiment 20. The system of any one of embodiments 1-19, wherein the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinant pathogen polypeptides indicates an infection from a pathogen in the subject.

[0022] Embodiment 21, The system of any one of embodiments 1-19, wherein the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinant pathogen polypeptides indicates vaccination against a pathogen in the subject.

[0023] Embodiment 22. The system of any one of embodiments 18-21, wherein the pathogen comprises a Bartonella species.

[0024] Embodiment 23. The system of any one of embodiments 18-22, further comprising a therapeutic regimen for treatment against the infection from the pathogen or a vaccine for protection against the pathogen.

[0025] Embodiment 24. The system of embodiment 20, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity of detecting the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide.

[0026] Embodiment 25. The system of embodiment 24, wherein the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0027] Embodiment 26. The system of embodiment 21, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity' of detecting the infection in the subject compared to a second sensitivity of detecting the infection in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide.

[0028] Embodiment 27. The system of embodiment 26, wherein the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0029] Embodiment 28. The system of embodiment 21, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the specificity of detecting the vaccination in the subject compared to a second specificity of detecting theAttorney Docket No.10620-163W01vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide.

[0030] Embodiment 29. The system of embodiment 28, wherein the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0031] Embodiment 30. The system of embodiment 21, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the sensitivity of detecting infected versus vaccinated subjects.

[0032] Embodiment 31. The system of embodiment 21, wherein the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0033] Embodiment 32. The system of any one of embodiments 1-31, wherein the antibody comprises a canine antibody.

[0034] Embodiment 33. The system of embodiment 32, wherein the antibody consists of the canine antibody.

[0035] Embodiment 34. The system of any one of embodiments 1-31, wherein the antibody comprises a human antibody.

[0036] Embodiment 35. The system of embodiment 34, wherein the antibody consists of the human antibody.

[0037] Embodiment 36. A kit comprising: two or more recombinant pathogen polypeptides selected from a group consisting of a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); an immunoglobulin binding agent and; an instruction manual.

[0038] Embodiment 37. The kit of embodiment 36, wherein the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains.

[0039] Embodiment 38. The kit of embodiment 36, wherein the two or more recombinant pathogen polypeptides comprise two or more immunodominant amino acid sequences.

[0040] Embodiment 39. The kit of any one of embodiments 36-38, wherein the two or more recombinant pathogen polypeptides are attached to a solid support.Attorney Docket No.10620-163W01

[0041] Embodiment 40. The kit of any one of embodiments 36-39, wherein the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides.

[0042] Embodiment 41, The kit of embodiment 40, wherein the two or more recombinant pathogen polypeptides comprise two or more Bartonella species polypeptides.

[0043] Embodiment 42. The kit of any one of embodiments 36-41, wherein the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15.

[0044] Embodiment 43. The kit of embodiment 42, wherein the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15.

[0045] Embodiment 44. The kit of any one of embodiments 36-43, further comprising a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding among: an antibody; the two or more recombinant pathogen polypeptides; and the immunoglobulin binding agent.

[0046] Embodiment 45. The kit of embodiment 44, wherein the signal moiety is complexed with the immunoglobulin binding agent.

[0047] Embodiment 46. The kit of embodiment 44, wherein the signal moiety comprises an enzyme.

[0048] Embodiment 47. The kit of embodiment 46, wherein the enzyme comprises a horseradish peroxidase (HRP).

[0049] Embodiment 48. The kit of embodiment 44, wherein the signal moiety comprises a fluorescent molecule.

[0050] Embodiment 49. The kit of any one of embodiments 36-48, further comprising a therapeutic regimen for treating an infection from a pathogen or a vaccine for protection against the pathogen.

[0051] Embodiment 50. The kit of embodiment 49, wherein the pathogen comprises a Bartonella species,

[0052] Embodiment 51. A method comprising: capturing an antibody in a sample obtained from a subject by contacting the sample with two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-0 (rATP-P), a recombinant GroEL (rGroEL), a recombinant Lem A (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5Attorney Docket No.10620-163W01(rVirB5), wherein the antibody binds to binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5).

[0053] Embodiment 52. A method comprising: (a) contacting a sample obtained from a subject with two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-β (rATP-β), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5); and (b) detecting binding between an antibody in the sample and the two or more recombinant pathogen polypeptides.

[0054] Embodiment 53. The method of embodiment 51 or 52, further comprising binding the antibody with an immunoglobulin binding agent.

[0055] Embodiment 54. The method of embodiment 53, wherein the immunoglobulin binding agent binds to an IgG.

[0056] Embodiment 55. The method of embodiment 54, wherein the IgG comprises IgGl, IgG2, IgG3, or IgG4.

[0057] Embodiment 56. The method of embodiment 53, wherein the immunoglobin binding agent is complexed with a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of a binding between the antibody and the two or more recombinant pathogen polypeptides.

[0058] Embodiment 57. The method of embodiment 56, wherein the signal moiety comprises an enzyme.

[0059] Embodiment 58. The method of embodiment 57, wherein the enzyme comprises a horseradish peroxidase (HRP)

[0060] Embodiment 59. The method of embodiment 56, wherein the signal moiety comprises a fluorescent molecule.

[0061] Embodiment 60. The method of any one of embodiments 51-59, wherein the antibody comprises an endogenous antibody.

[0062] Embodiment 61. The method of any one of embodiments 51-60, wherein the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains.

[0063] Embodiment 62. The method of any one of embodiments 51-61, wherein the two or more recombinant pathogen polypeptides comprise two or more immunodominant amino acid sequences.Attorney Docket No.10620-163W01

[0064] Embodiment 63. The method of any one of embodiments 51-62, wherein the two or more immunodominant amino acid sequences are selected based on B-cell epitope mapping, betaturn, surface accessibility, antigenicity, hydrophilicity prediction, or a combination thereof.

[0065] Embodiment 64. The method of any one of embodiments 51-63, wherein the two or more recombinant pathogen polypeptides are attached to a solid support.

[0066] Embodiment 65. The method of any one of embodiments 51-64, wherein the two or more recombinant pathogen polypeptides comprise two or more Bartonella species polypeptides.

[0067] Embodiment 66. The method of any one of embodiments 51-63, wherein the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15.

[0068] Embodiment 67. The method of embodiment 66, wherein the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15.

[0069] Embodiment 68, The method of any one of embodiments 51-67, wherein the antibody is produced in response to an infection from a pathogen in the subject.

[0070] Embodiment 69. The method of embodiment 68, wherein detecting the antibody in the sample binding to the two or more recombinant pathogen polypeptides indicates the infection from the pathogen in the subject.

[0071] Embodiment 70, The method of any one of embodiments 51-67, wherein the antibody is produced in response to vaccination against a pathogen in the subject.

[0072] Embodiment 71. The method of embodiment 70, wherein detecting the antibody in the sample binding to the two or more recombinant pathogen polypeptides indicates vaccination against the pathogen in the subject.

[0073] Embodiment 72. The method of any one of embodiments 68-71, wherein the pathogen comprises a Bartonella species.

[0074] Embodiment 73. The method of embodiment 68 or 69, further comprising treating the subject infected with the pathogen.

[0075] Embodiment 74. The method of embodiment 70 or 71, further comprising protecting the subject against the pathogen.

[0076] Embodiment 75. The method of embodiment 68 or 69, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity for detectingAttorney Docket No.10620-163W01the infection in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide.

[0077] Embodiment 76. The method of embodiment 75, wherein the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0078] Embodiment 77. The method of embodiment 68 or 69, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity of detecting the infection in the subject compared to a second sensitivity of detecting the infection in the subject, wherein the second sensitivity is obtained from detecting binding between the antibody and one pathogen polypeptide.

[0079] Embodiment 78. The method of embodiment 77, wherein the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0080] Embodiment 79. The method of embodiment 70 or 71, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the vaccination in the subject compared to a second specificity for detecting the vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide.

[0081] Embodiment 80. The method of embodiment 79, wherein the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0082] Embodiment 81, The method of embodiment 70 or 71, wherein detecting binding between the antibody and two or more recombinant pathogen polypeptides increases the sensitivity of detecting infected versus vaccinated subjects.

[0083] Embodiment 82. The method of embodiment 81, wherein the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

[0084] Embodiment 83. The method of any one of embodiments 51-82, wherein the antibody comprises a canine antibody.

[0085] Embodiment 84. The method of embodiment 83, wherein the antibody consists of the canine antibody.Attorney Docket No.10620-163W01

[0086] Embodiment 85. The method of any one of embodiments 51-82, wherein the antibody comprises a human antibody.

[0087] Embodiment 86. The method of embodiment 85, wherein the antibody consists of the human antibody.

Claims

Attorney Docket No.10620-163W01CLAIMSWe claim:

1. A composition comprising two or more recombinant pathogen polypeptides selected from a group consisting of: a recombinant ATP-0 (rATP-P), a recombinant GroEL (rGroEL), a recombinant LemA (rLemA), a recombinant SucB (rSucB), and a recombinant VirB5 (rVirB5).

2. The composition of claim 1, wherein the two or more recombinant pathogen polypeptides comprise two or more cytoplasmic domains.

3. The composition of claim 1 or 2, wherein the two or more recombinant pathogen polypeptides comprise immunodominant amino acid sequences.

4. The composition of claim 3, wherein the two or more immunodominant amino acid sequences are selected based on B-cell epitope mapping, beta-turn, surface accessibility, antigenicity, hydrophilicity prediction, or a combination thereof.

5. The composition of any one of claims 1-4, wherein the two or more recombinant pathogen polypeptides are attached to a solid support.

6. The composition of any one of claims 1-5, wherein the two or more recombinant pathogen polypeptides comprise two or more Bartonella species pathogen polypeptides.

7. The composition of claim 1, wherein the two or more recombinant pathogen polypeptides are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any two or more of SEQ ID NOs: 1-15.

8. The composition of claim 7, wherein the two or more recombinant polypeptides are identical to any two or more of SEQ ID NOs: 1-15.

9. The composition of claim 1, comprising recombinant ATP-β and recombinant GroEL.

10. A system comprising:(a) the composition of any one of claims 1-9; and(b) an immunoglobulin binding agent, wherein the immunoglobin binding agent binds to an antibody, wherein the antibody binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5).

11. The system of claim 10, wherein the immunoglobulin binding agent binds to a IgG.Attorney Docket No.10620-163W0112. The system of claim 11, wherein the IgG comprises IgGl, IgG2, IgG3, or IgG4.

13. The system of any one of claims 10-12, further comprising a signal moiety, wherein detection of a signal generated by the signal moiety is indicative of binding between the antibody and the two or more recombinant pathogen polypeptides.

14. The system of claim 13, wherein the signal moiety is complexed with the immunoglobulin binding agent.

15. The system of claim 13, wherein the signal moiety comprises an enzyme.

16. The system of claim 15, wherein the enzyme comprises a horseradish peroxidase (HRP).

17. The system of claim 13, wherein the signal moiety comprises a fluorescent molecule.

18. The system of any one of claims 10-17, wherein the antibody comprises an endogenous antibody.

19. The system of any one of claims 10-18, wherein the antibody binds to two or more of the recombinant pathogen polypeptides.

20. The system of any one of claims 10-19, wherein the antibody is produced in response to an infection from a pathogen.

21. The system of any one of claims 10-19, wherein the antibody is produced in response to vaccination against a pathogen.

22. The system of any one of claims 10-21, wherein the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinant pathogen polypeptides indicates an infection from a pathogen in the subject.

23. The system of any one of claims 10-21, wherein the antibody is present in a sample obtained from a subject, and wherein the antibody binding to the two or more recombinant pathogen polypeptides indicates vaccination against a pathogen in the subject.

24. The system of any one of claims 20-23, wherein the pathogen comprises a Bartonella species.

25. The system of any one of claims 20-24, further comprising a therapeutic regimen for treatment against the infection from the pathogen or a vaccine for protection against the pathogen.

26. The system of claim 22, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a specificity for detecting the infection in the subject compared to a second specificity of detecting the infection in the subject, wherein theAttorney Docket No.10620-163W01second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide.

27. The system of claim 26, wherein the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

28. The system of claim 23, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases a sensitivity of detecting the infection in the subject compared to a second sensitivity of detecting the infection in the subject, wherein the second sensitivity' is obtained from detecting binding between the antibody and one pathogen polypeptide.

29. The system of claim 28, wherein the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

30. The system of claim 23, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the specificity of detecting the vaccination in the subject compared to a second specificity of detecting the vaccination in the subject, wherein the second specificity is obtained from detecting binding between the antibody and one pathogen polypeptide.

31. The system of claim 30, wherein the specificity compared to the second specificity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

32. The system of claim 23, wherein detecting binding between the antibody and the two or more recombinant pathogen polypeptides increases the sensitivity of detecting infected versus vaccinated subjects.

33. The system of claim 23, wherein the sensitivity compared to the second sensitivity is increased by at least 5%, at least 10%, at least 20%, at least 50%, at least 100%, or at least 500%.

34. The system of any one of claims 10-33, wherein the antibody comprises a canine antibody.

35. The system of claim 34, wherein the antibody consists of the canine antibody.

36. The system of any one of claims 10-35, wherein the antibody comprises a human antibody.

37. The system of claim 36, wherein the antibody consists of the human antibody.

38. A kit comprising:(a) the composition of any one of claims 1-9;Attorney Docket No.10620-163W01(b) an immunoglobulin binding agent; and(c) an instruction manual.

39. A method comprising:capturing an antibody in a sample derived from a subject by contacting the sample with the composition of any of claims 1-9 or the system of any of claims 10-37,wherein the antibody binds to any one of the recombinant ATP-β (rATP-β), the recombinant GroEL. (rGroEL), the recombinant LemA (rLemA), the recombinant SucB (rSucB), and the recombinant VirB5 (rVirB5).

40. A method comprising:contacting a sample derived from a subject with the composition of any of claims 1-9 or the system of any of claims 10-37; anddetecting binding between an antibody in the sample and the two or more recombinant pathogen polypeptides.

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