Babesia Biomarkers for Diagnostic and Screening In Vitro Diagnostic Test
20180372742 ยท 2018-12-27
Inventors
- Douglas Molina (Buena Park, CA, US)
- Arlo Randall (Huntington Beach, US)
- Gary Hermanson (Encinitas, CA, US)
- Choukri Ben Mamoun (Irvine, CA, US)
Cpc classification
G01N2469/20
PHYSICS
International classification
Abstract
An antigen composition with plurality of antigens from B. microti, and methods of predicting a likelihood of an individual having B. microti infection using the antigen composition as biomarkers of infections of B. microti to human are provided. In particularly preferred aspects, the antigens are immunodominant and have quantified and known relative reactivities with respect to sera of a population infected with the pathogen with an average discriminatory p value of 0.05.
Claims
1. An antigen composition comprising: a plurality of antibody reactive antigens associated with a carrier, wherein at least two of the antigens have quantified and known relative antibody reactivities with respect to sera of a population infected by Babesia microti; and wherein the at least one of the plurality of antigens are selected from the group consisting of BBM_03g00785, BBM_03g04695, BBM_01g00985, BBM_03g00947, BBM_03g00960, BBM_03g04695, BBM_03g02345, BBM_04g09980, BBM_03g03480, BBM_04g06560, BBM_02g04285, BBM_03g02390, BBM_02g04265, BBM_04g05080, BBM_03g00020, BBM_02g02608, BBM_02g04280, BBM_02g04260, BBM_02g03140, BBM_02g00896, BBM_03g03430, BBM_02g00010, BBM_02g04275, BBM_03g00790, BBM_02g02940, BBM_04g07556, BBM_04g07810, BBM_02g04060, BBM_04g06750, BBM_01g01510, BBM_02g03960, BBM_01g02740, BBM_02g04250, BBM_01g02740, BBM_04g05345, BBM_03g02840, BBM_02g00005, BBM_01g02415, BBM_03g04760, BBM_01g02545, BBM_03g01870, BBM_03g04565, BBM_04g09230, BBM_02g02185, BBM_04g07915, BBM_04g08970, BBM_02g03947, BBM_04g09575, BBM_01g00590, BBM_03g01960, BBM_02g02960. BBM_04g07920, BBM_03g01540, BBM_03g03675, BBM_04g09765, or fragments thereof.
2. The antigen composition of claim 1, wherein the known reactivities are characterized by strength of reactivity.
3. The antigen composition of claim 1, wherein the at least one of the plurality of antigens have an average discriminatory p value of 0.05.
4. The antigen composition of claim 1, wherein the known relative antibody reactivities are IgG reactivities or IgM reactivities
5. (canceled)
6. The antigen composition of claim 1, wherein the known relative antibody reactivities are at least 30% higher compared to with respect to sera of a population uninfected by Babesia microti.
7. (canceled)
8. The antigen composition of claim 1, wherein the carrier is a solid carrier, and the plurality of antigens is disposed on the solid carrier in an array.
9. (canceled)
10. The antigen composition of claim 1, wherein antibodies to the at least two antigens are present in at least 60% of a population infected by Babesia microti.
11. A method of predicting a likelihood of an individual having Babesia microti infection, comprising: providing a serum sample of the individual; reacting the serum sample with a plurality of antigens antibody reactive antigens associated with a carrier, wherein at least two of the antigens have quantified and known relative antibody reactivities with respect to sera of a population infected by Babesia microti; wherein the at least one of the plurality of antigens are selected from the group consisting of BBM_03g00785, BBM_03g04695, BBM_01g00985, BBM_03g00947, BBM_03g00960, BBM_03g04695, BBM_03g02345, BBM_04g09980, BBM_03g03480, BBM_04g06560, BBM_02g04285, BBM_03g02390, BBM_02g04265, BBM_04g05080, BBM_03g00020, BBM_02g02608, BBM_02g04280, BBM_02g04260, BBM_02g03140, BBM_02g00896, BBM_03g03430, BBM_02g00010, BBM_02g04275, BBM_03g00790, BBM_02g02940, BBM_04g07556, BBM_04g07810, BBM_02g04060, BBM_04g06750, BBM_01g01510, BBM_02g03960, BBM_01g02740, BBM_02g04250, BBM_01g02740, BBM_04g05345, BBM_03g02840, BBM_02g00005, BBM_01g02415, BBM_03g04760, BBM_01g02545, BBM_03g01870, BBM_03g04565, BBM_04g09230, BBM_02g02185, BBM_04g07915, BBM_04g08970, BBM_02g03947, BBM_04g09575, BBM_01g00590, BBM_03g01960, BBM_02g02960. BBM_04g07920, BBM_03g01540, BBM_03g03675, BBM_04g09765, or fragments thereof.
12. The method of claim 11, wherein the plurality of the antigens have a known association with a disease parameter, wherein the disease parameter is selected from the group consisting of a previous or current exposure to a pathogen, acute infection with a pathogen, latent or recurrent infection with a pathogen, and at least partial immunity to infection with a pathogen.
13. The method of claim 11, wherein the carrier is a pharmaceutically acceptable carrier, and wherein the composition is formulated as a vaccine.
14. The method of claim 11, wherein the carrier is a solid carrier, and the plurality of antigens is disposed on the carrier in an array.
15. The method of claim 11, wherein at least one of the plurality of antigens or fragments thereof are recombinant.
16. The method of claim 11, wherein the plurality of antigens or fragments thereof are at least partially purified.
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. A diagnostic kit for testing Babesia microti infection in patients suspected to have Babesia microti infection, comprising: a plurality of antibody reactive antigens associated with a carrier, wherein at least two of the antigens have quantified and known relative antibody reactivities with respect to sera of a population infected by Babesia microti; and wherein the at least one of the plurality of antigens are selected from the group consisting of BBM_03g00785, BBM_03g04695, BBM_01g00985, BBM_03g00947, BBM_03g00960, BBM_03g04695, BBM_03g02345, BBM_04g09980, BBM_03g03480, BBM_04g06560, BBM_02g04285, BBM_03g02390, BBM_02g04265, BBM_04g05080, BBM_03g00020, BBM_02g02608, BBM_02g04280, BBM_02g04260, BBM_02g03140, BBM_02g00896, BBM_03g03430, BBM_02g00010, BBM_02g04275, BBM_03g00790, BBM_02g02940, BBM_04g07556, BBM_04g07810, BBM_02g04060, BBM_04g06750, BBM_01g01510, BBM_02g03960, BBM_01g02740, BBM_02g04250, BBM_01g02740, BBM_04g05345, BBM_03g02840, BBM_02g00005, BBM_01g02415, BBM_03g04760, BBM_01g02545, BBM_03g01870, BBM_03g04565, BBM_04g09230, BBM_02g02185, BBM_04g07915, BBM_04g08970, BBM_02g03947, BBM_04g09575, BBM_01g00590, BBM_03g01960, BBM_02g02960. BBM_04g07920, BBM_03g01540, BBM_03g03675, BBM_04g09765, or fragments thereof.
23. The test kit of claim 22, wherein the known reactivities are characterized by strength of reactivity.
24. The test kit of claim 22, wherein the at least one of the plurality of antigens have an average discriminatory p value of0.05.
25. The test kit of claim 22, wherein the known relative antibody reactivities are IgG reactivities or IgM reactivities
26. (canceled)
27. The test kit of claim 22, wherein the known relative antibody reactivities are at least 30% higher compared to with respect to sera of a population uninfected by Babesia microti.
28. (canceled)
29. The test kit of claim 22, wherein the carrier is a solid carrier, and the plurality of antigens is disposed on the solid carrier in an array.
30. (canceled)
31. The test kit of claim 22, wherein antibodies to the at least two antigens are present in at least 60% of a population infected by Babesia microti.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0018]
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[0020]
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[0028]
DETAILED DESCRIPTION
[0029] In the present application, the inventors identified a group of antigens that shows relatively high reactivities to IgG and/or IgM of an infected population to B. microti. At least some of these antigens are highly specific and sensitive such that they are suitable to be used as biomarkers of B. microti infection.
[0030] The present inventive subject matter provides for the identification, analysis, and monitoring of antibody reactivity to specific B. microti antigens, or antigen sets, which has diagnostic, prognostic, and therapeutic value, specifically with respect to various diseases, disorders, and symptoms related to B. microti infection. The present inventive subject matter also provides tools and methods to accurately survey B. microti infections, disorders, and diseases via the combination of antigen/antibody reactivity detection and monitoring, and characterizing sera samples of uninfected and infected by B. microti.
[0031] It should be noted that in the following description antigens may be identified by either the gene descriptor for the gene that encodes the protein antigen and/or the name for the protein antigen. Thus, it should be understood that where the context indicates that a sequence or antigen is a protein sequence, a gene name for that sequence or antigen denotes the protein product for that gene. Where reference is made to antibodies it is recognized that while such antibodies may be referred to as having been obtained from serum, said antibodies may also derived from other sources, including, but not limited to, mucus, saliva, semen, lacrimal fluid, urine, feces, aqueous humor, pus, cerebrospinal fluid, lymphatic fluid, and synovial fluid.
[0032] The present inventive subject matter is also directed to the identification of specific B. microti antigens that trigger antibody reactivity associated with various diseases, disorders, or symptoms related to B. microti infection, which the specific antigens have predetermined antibody reactivities from serum of a population of B. microti Infected patients. Thus, such specific antigens may have a statistically high probability to elicit antibody responses in a relatively large group of B. microti infected patients.
[0033] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0034] In some embodiments, the numbers expressing quantities or ranges, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term about. Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Not withstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints and open-ended ranges should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.
[0035] As used in the description herein and throughout the claims that follow, the meaning of a, an, and the includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of in includes in and on unless the context clearly dictates otherwise.
[0036] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0037] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.
[0038] One aspect of the invention includes an antigen composition that is specific and sensitive to be a biomarker of B. microti infection. The antigen composition includes a plurality of antibody (IgG, IgM) reactive antigens. At least two of these antigens have quantified and known relative antibody reactivities with respect to sera of a population infected by Babesia microti. It is highly preferred that among these antigens, at least one antigen has an average discriminatory p value of 0.05.
[0039] The biomarker antigens of B. microti infection are identified from a proteome screen against sera of a population that has been previously exposed to the B. microti, which can be compared with sera of a population that has not been exposed to the B. microti. It is preferred that the screening also provides data of relative reactivities with respect to the antigens and sera of the populations
[0040] While not limiting to the inventive subject matter, in a preferred embodiment, the plurality of antigens are selected as a sampling of the B. microti secretome, GPI-anchored proteome, the metabolic proteome and other proteins. The secretome is the totality of secreted organic molecules and inorganic elements by biological cells, tissues, organs, and organisms. The GPI-anchored proteome is the totality of surface proteins anchored to the cell membrane via a GPI anchor. Because secreted proteins and surface proteins are naturally exposed to outside of the infecting organism (B. microti) and can be detected by antibodies in the infected organisms' sera, the GPI-anchored proteome and secretome are considered useful as antigens for diagnosing pathogen infections.
[0041] It is generally preferred that at least part of the B. microti's genome is obtained and all potential open reading frames and portions thereof are determined in silco. Once the potential genes are identified, suitable primers are determined to provide amplicons of the entire Open Reading Frames (ORFs), or, less preferably, portions thereof, when in the primers are preferably designed to allow facile subcloning into an expression system. Most preferably, the subcloning uses recombinase-based subcloning using unpurified PCR mixtures to avoid cloning bias, and the so obtained recombinant plasmids are polyclonally multiplied, which enables unbiased presentation of the amplicons. It is still further particularly preferred that the plasmid preparations are then subjected to an in vitro transcription/translation reaction to thereby provide the recombinant ORF peptide, which is then spotted or otherwise immobilized onto a suitable addressable carrier (e.g., membrane, bead, etc.).
[0042] It should be recognized that the so prepared proteomes can then be exposed to serum of a population of control individuals and/or population of individuals that are known to have exposure to B. microti from which the ORFs were prepared. Antibodies of the serum that bind to one or more of the ORFs are then detected using well known methods (e.g., use of secondary antibodies etc.). In this manner, the entire proteome of the pathogen can be rapidly assessed for immunogenicity and potential binding with antibodies in serum. Various preferred aspects, compositions, and methods of proteome preparation are disclosed in International patent publication number WO 06/088492, which is incorporated by reference herein.
[0043] It should be also noted that not all individuals show same response or development of antibodies to an antigen when they are exposed (or infected) by the antigen. Such individual differences in immune responses are, at least in part, due to their variability and specificity of major histocompatibility complex (MHC) types. Thus, an antigen that can be highly immunogenic to individual A may not be immunogenic to individual B. That being said, it is virtually impossible to predict whether a protein from a pathogen would be an immunogenic to human being by simply determining the types of the protein. Consequently, it is also virtually impossible to predict or determine an antigen or a set of antigens simply by looking at their protein sequences or genomic sequences whether such antigens would be immunogenic to human population. Therefore, and among various other advantages, it should be especially recognized that contemplated compositions and methods presented herein will allow for preparation of diagnostic compositions comprising a plurality of antigens with known and predetermined affinity to target ORFs of B. microti. As individual immune systems are known to exhibit significant variation with respect to antigen recognition, methods and compositions contemplated herein will allow statistically supported antigen identification to identify immunodominant antigens in a population of infected patient. Consequently, multiple targets can be used to elicit an immune response and/or detect a prior exposure, even where one or more of the targets may be evasive for detection or provide only a weak response.
[0044] With respect to the immunodominant sequences identified herein, it should be further appreciated that the sequences need not be complete ORFs, but that suitable sequences may also be partial sequences (e.g., synthetic, recombinant or isolated, etc.) that typically comprise at least part of an antigenic epitope. For example, contemplated DNA sequences include those that will hybridize under stringent hybridization conditions to respective sequences listed in the sequence listing. Thus, sequences contemplated herein may be identified as DNA sequences encoding the antigenic peptide (partial or entire ORF), or may be identified as peptide sequence (or homologs thereof). Similarly, chemically modified antigens, and/or orthologs of the polypeptides presented herein are also deemed suitable for use herein.
[0045] It should be particularly noted that while proteome screening will provide a plurality of antigens as potentially useful molecules for diagnosis, vaccination, and/or therapy, such an approach only provides a raw cut of (a plurality) of individual responses. Therefore, as most individual immune reactions towards the same pathogen elicit a significantly distinct profile of antibodies (e.g., depending on disease stage, previous exposure, and/or inter-individual variability), results obtained from such screening are typically inhomogeneous. Consequently, variability of the individual immune responses and variability of the quantity of recombinant protein in the array must be taken into consideration to obtain meaningful results.
[0046] Therefore, it should be appreciated that filtering of raw data will result in a collection of antigens with quantified and known relative reactivities with respect to sera of a population infected with B. microti. Moreover, it should be noted that as signals may be specific to a particular stage in the course of an infection, relative reactivities may be indicative of the time course of the infection, and/or relative reactivities may represent differences in the strength of immunogenicity of the particular antigen (or quantity of deposited antigen in the screening assay). Additionally, it should be particularly recognized that depending on the choice of the specific patient population, the tested sera will reflect the immune status of a population that is characterized by one or more parameters of the disease. For example, populations may be observed that are infected or not infected, that had a long-term exposure or chronic infection, which had spontaneous recovery, that represents a group of responders (or non-responders) to a particular drug treatment, or that had at least partial immunity to the pathogen.
[0047] In still further contemplated aspects, immunodominant antigens are identified by selecting for an antigen that (a) produces in at least 50% of a population a measurable signal, preferably in at least 60% of a population, and (b) has a signal strength of at least 40%, preferably at least 50% of the overall average signal intensity. However, and more preferably, the signal strength will be at least above average of the overall average signal intensity, and even more preferably in the upper tertile (quartile, or even quintile) of sizial intensities in the assay. Still further, it is generally preferred that the series of tests also include a negative control against which the potential immunodominant antigens are compared. Alternatively, immunodominant antigens can be identified by selecting for an antigen that shows high average discriminatory power and as such have a t-test p-value of 0.05, an area under the curve (AUC) of >0.75, and most preferably a Benjamini-Hochberg corrected p-value of 0.05.
[0048] Consequently, and with particular respect to the pathogen (B. microti) presented herein, it should be appreciated that compositions comprising one or more selected immunodominant antigens can be prepared that will have a statistically high probability to elicit or have elicited an immune response in a relatively large group of patients. Each of the antigens was characterized, inter alia, with regard to their individual and relative reactivities for the pathogen. Most typically, reactivity was measured as strength of immunogenicity (e.g., such that average binding affinity and/or average quantity of the antibodies produced a predetermined signal intensity (e.g., in the upper half, upper tertile, or even upper quartile)). In a preferred embodiment, the relative reactivities are quantified based on the antigens' reactivities to IgG. In some other embodiments, the relative reactivities are quantified based on the antigens' reactivities to IgM. In still some other embodiments, the relative reactivities are quantified based on the antigens' reactivities both to IgG and IgM. Further, it is contemplated that the relative reactivities of antigens suitable to be selected as biomarkers of B. microti infection are at least 30% higher, preferably at least 50%, compared to with respect to sera of a population uninfected by B. microti.
[0049] Preferably, the antigens used as biomarkers have high average discriminatory power and as such have a t-test p-value of 0.05, an area under the curve (AUC) of >0.75, and most preferably a Benjamini-Hochberg, corrected p-value of 0.05.
[0050] Selected antigens as biomarkers can be associated with a carrier. In some embodiments, carriers can be solid carriers (e.g., a plate, a membrane, a bead, a chip, a microfluidic device, etc.). In other embodiments, carriers can be non-solid carriers (e.g., liquid carriers, gas carriers, etc.). In the embodiments where the carriers are solid carriers, it is preferred that the antigens are deposited in an array. For example, antigens can be arrayed on a chip that can be read in an automated device (e.g., via scanner) or visual manner (e.g., dye-forming colorimetric reaction, etc.). Most typically, in these embodiments, the plurality of antigens is deposited in a spatially addressable manner (e.g., x-y matrix or beads with color association or microtiter plate, etc.). Moreover, it should be noted that diagnostic devices contemplated herein may be based on numerous well known manners of detection, including ELISA (sandwich or non-sandwich), competitive ELISA, anti-idiotypic antibodies, etc., wherein all known colorimetric and photometric (e.g., fluorescence, luminescence, etc.) or radiometric reactions are deemed suitable for use.
[0051] More specifically, in these embodiments, a plurality of immunodominant antigens of B. microti are deposited on a solid surface or onto an addressable solid phase and exposed to blood, serum, plasma or other antibody-containing body fluid. Consequently, so prepared compositions can be employed to identify and/or characterize an immune response of an individual against selected antigens, and optionally assess the kind of immune response (e.g., identification of latent or chronic infection, etc.), as well as disease progression, efficacy of therapy, etc. Most typically, the plurality of antigens will include between 5 to 10 antigens, but significantly higher amounts of antigens are also contemplated, including at least 25%, more typically at least 50%, even more typically at least 75%, and most typically at least 90% of the proteose of the pathogen. Similarly, less than 5 antigens (1-4) are also deemed suitable. In further typical aspects of the inventive subject matter, contemplated arrays are most preferably processed in a microfluidic device. For example, an array of antigens in such devices may be printed on a membrane or other material (e.g., nitrocellulose-coated carrier of less than 1 cm.sup.2 area) that is then placed in a microfluidic device having sample/reagent inlet and outlet ports. Depending on the specific configuration, signals may be acquired using optical methods (e.g., charge coupled device (CCD) chip, flat bed scanner, etc.), electrical methods (e.g., voltametric or amperometric, etc.), or other methods well known in the art. Alternatively, visual detection or detection using a regular flat bed scanner at 1200 dpi resolution and/or fluorescence detection is also deemed suitable.
[0052] It is contemplated that antibodies against at least some immunodominant antigens may block invasion of B. microti to red blood cells. Thus, those immunodominant antigens can be effective therapeutic compositions or vaccines to B. microti infection. Thus, in another aspect of the inventive subject matter, the carrier is a pharmaceutically acceptable carrier, and the one or more immunodominant antigens are formulated as a vaccine or therapeutic compositions. In such aspects, it is generally preferred that the vaccine comprises multiple (e.g., at least two, four, or six, etc.) antigens. Depending on the antigens, it is contemplated that the antigens or fragments thereof are at least partially purified and/or recombinant.
EXAMPLES
[0053] The inventors have identified biomarkers to include in a test that are detected by IgG and/or IgM antibodies to overcome the challenges of early detection of B. microti infection. Further, the inventors have used genomics and statistical analysis to clone high priority targets and tested them all for diagnostic utility. More importantly, the inventors identified at least 54 B. microti proteins that can be used alone or in combination to develop an in vitro diagnostic (IVD) for Babesiosis and a blood screening test for TTB.
[0054] In these examples, the inventors interrogated the targeted B. microti protein array with at least 34 human samples with known B. microti exposure, at least 60 wild mice with known immunofluorescence antibody assay (IFA) titers and at least 4 experimentally infected mice.
[0055]
[0056] In
[0057]
[0058] The inventors also tested mouse sera to identify the epitopes that may be conserved because they have little to no evolutionary pressure to change.
[0059] The inventors also tested the IgG and IgM reactivities in wild mice infected with B. microti using B. microti protein arrays used in
[0060] Table 1 shows statistical immunogenicity data of a set of B. microti antigens to human IgG and IgM. Specifically, each antigen is tested using a plurality of human serum infected or uninfected by B. microti. The IgG and IgM antibody titers were measured and mean values of its titers among B. microti infected population (mean positive value) and uninfected population (mean negative value) were presented. Generally, higher positive antibody titer (higher positive value) to an antigen may indicate higher immunogenicity or reactivity of the antigen. To eliminate the false negative or false positive reactivities of some antigens, discriminatory power values were also calculated using antibody titers of infected and uninfected population, including a t-test p-value and an area under the curve (AUC) for each antigen for IgG and IgM. Generally, an at that shows higher discriminatory power values (e.g., t-test p-value<0.1, preferably, t-test p-value<0.05, more preferably, t-test p-value<0.01, an or AUC>0.75, preferably AUC>0.85, more preferably AUC>0.95, etc.) indicates stronger relative reactivities of the antigen. Thus, relative reactivities of antigens can be determined by one or more statistical value indicating discriminatory power values of such antigens.
TABLE-US-00001 TABLE 1 Human IgG Antigen.ID P.Value AUC Mean.Neg Mean.Pos Human IgM BBM_03g00785.3029 8.99E10 0.95 0.399293 4.789639 BBM_03g04695_s1.661 3.05E09 0.93 0.669698 5.481812 BBM_01g03280.2214 9.63E09 0.94 2.456043 6.449811 BBM_03g00947.3924 8.58E09 0.95 0.275067 3.86978 BBM_04g06170.112 0.00239133 0.8 1.196694 2.81381 BBM_04g07360.2433 6.77E11 0.94 0.755438 5.648211 BBM_01g01305.3364 0.00022647 0.84 2.68284 4.747776 BBM_03g01350.3631 1.33E05 0.85 1.046079 3.35897 BBM_01g03430.1210 0.8555205 0.46 5.223108 5.140795 BBM_01g00150.2961 0.9056553 0.47 4.576688 4.524298 BBM_04g06785.4411 0.01999478 0.72 2.77311 4.087223 BBM_03g00820.2139 5.81E07 0.9 1.587474 4.719381 BBM_04g05780.3646 0.34480926 0.64 2.238249 2.70729 BBM_02g01420.4098 0.89471116 0.48 4.015039 3.954798 BBM_02g01611.3913 7.86E03 0.9 0.3352 0.78502 BBM_04g09840.4118 0.90989476 0.47 4.460904 4.409516 BBM_03g00020.2501 0.00035712 0.87 0.496188 2.351402 BBM_01g01620_s1.2140 0.00183958 0.77 1.598854 2.947658 BBM_03g00840.3233 0.87324552 0.46 3.98483 3.914705 BBM_03g03976.3506 0.92943889 0.48 4.802056 4.761534 BBM_01g00590.384 2.02E05 0.91 0.541924 3.003248 BBM_01g00500.3846 0.08659995 0.68 1.178904 2.111308 BBM_02g02170.2365 0.91005807 0.48 4.892041 4.840592 BBM_02g00910.2993 0.37235784 0.46 2.349886 1.815883 BBM_03g00452.1539 0.00334588 0.88 0.114319 0.927211 BBM_01g01135.421 0.04666509 0.68 1.805466 2.996336 BBM_04g09615_e1of2_s1of1.3781 0.09067509 0.67 1.550336 2.364191 BBM_01g00985_s2.2509 1.84E06 0.9 0.842178 3.922386 BBM_01g00985_s1.3665 7.70E07 0.9 1.272292 4.523013 BBM_03g00450.1002 0.9485982 0.58 1.840978 1.811501 BBM_03g04295.771 0.03438416 0.76 0.023603 0.364221 BBM_01g00435.596 0.72592556 0.56 1.679258 1.841831 BBM_03g04505.2457 0.07771156 0.67 0.781672 1.217634 BBM_01g00160.2425 0.00555469 0.77 0.445386 1.476276 BBM_03g01075_s2.4543 0.01491108 0.69 0.471006 0.992889 BBM_02g04265.3564 0.01011674 0.78 0.913018 2.288798 BBM_03g02305.2730 0.00090101 0.8 0.76321 2.316844 BBM_03g03046.750 0.00389917 0.75 1.790389 3.603164 BBM_03g01622_e1of3_s1of1.1464 0.00225706 0.84 0.58517 1.82656 BBM_02g00975.2145 0.00567154 0.77 0.318504 1.091411 BBM_02g00810_s2.800 0.00471435 0.77 0.11524 1.147255 BBM_04g08570.3912 0.4258462 0.67 0.514197 0.725369 BBM_03g01705_e1of5_s1of1.4356 0.00031077 0.84 0.376543 1.425722 BBM_04g07915_s1.3376 0.10883313 0.65 2.230289 3.294055 BBM_01g00985.2215 2.70E07 0.92 1.212289 4.288104 BBM_03g00960.4212 8.38E09 0.93 0.460178 2.947206 BBM_03g03925.1879 0.00011368 0.84 0.264361 1.584464 BBM_02g01875.2757 0.02031508 0.7 1.270701 2.372419 BBM_04g05680.1865 0.00104163 0.79 0.434877 1.578809 BBM_04g09295.3890 0.62386187 0.62 0.64532 0.8143 BBM_02g01015_e1of6_s1of1.4036 0.015301 0.82 0.12908 0.484692 BBM_03g02515_s1.431 0.0086695 0.71 1.129206 2.229231 BBM_03g00315_s2.3982 0.02945243 0.73 0.884862 1.799952 BBM_04g05260_s1.2831 0.03992059 0.7 2.035397 3.124443 BBM_03g02830.468 0.00070694 0.84 0.314615 1.432756 BBM_01g01800.1195 0.09548049 0.62 0.944946 1.616112 BBM_02g04285.2351 3.23E07 0.91 0.621857 4.25867 Human IgG P.Value AUC Mean.Neg Mean.Pos BBM_03g00785.3029 4.99E07 0.940476 0.095599 2.891633 BBM_03g04695_s1.661 4.11E06 0.910714 0.272967 2.256857 BBM_01g03280.2214 9.43E08 0.922619 0.664363 2.553439 BBM_03g00947.3924 3.34E05 0.91369 0.326227 2.165435 BBM_04g06170.112 0.000245 0.925595 0.247496 2.047512 BBM_04g07360.2433 8.43E07 0.916667 0.209352 2.006523 BBM_01g01305.3364 0.000203 0.866071 0.497504 2.098612 BBM_03g01350.3631 0.00024 0.854167 0.403865 1.950929 BBM_01g03430.1210 0.000344 0.842262 1.453502 2.821564 BBM_01g00150.2961 0.000386 0.842262 0.744639 2.093507 BBM_04g06785.4411 0.000349 0.848214 1.190192 2.520046 BBM_03g00820.2139 2.95E05 0.863095 0.444266 1.730086 BBM_04g05780.3646 0.001536 0.869048 0.432923 1.683829 BBM_02g01420.4098 0.00068 0.824405 0.714072 1.816276 BBM_02g01611.3913 0.001085 0.83631 0.30714 0.778514 BBM_04g09840.4118 0.001457 0.794643 1.038822 2.112978 BBM_03g00020.2501 0.004288 0.83631 0.186958 1.206587 BBM_01g01620_s1.2140 0.004634 0.770833 0.772469 1.782977 BBM_03g00840.3233 0.001287 0.797619 0.702094 1.705535 BBM_03g03976.3506 0.002931 0.732143 1.300336 2.295758 BBM_01g00590.384 0.000807 0.895833 0.24302 1.209739 BBM_01g00500.3846 0.002314 0.72619 0.30527 1.224276 BBM_02g02170.2365 0.003017 0.738095 1.27648 2.17413 BBM_02g00910.2993 0.000295 0.830357 0.183629 1.079076 BBM_03g00452.1539 0.000242 0.889881 0.01193 0.880743 BBM_01g01135.421 0.007734 0.848214 0.337546 1.21898 BBM_04g09615_e1of2_s1of1.3781 0.001632 0.821429 0.471247 1.344285 BBM_01g00985_s2.2509 0.000443 0.842262 0.119733 0.988987 BBM_01g00985_s1.3665 0.000489 0.827381 0.601772 1.45991 BBM_03g00450.1002 0.000435 0.821429 0.418348 1.269846 BBM_03g04295.771 0.004563 0.869048 0.001928 0.834462 BBM_01g00435.596 0.000649 0.860119 0.412148 1.229173 BBM_03g04505.2457 0.003029 0.827381 0.302568 1.11793 BBM_01g00160.2425 0.001725 0.833333 0.173807 0.979739 BBM_03g01075_s2.4543 0.012213 0.809524 0.205262 1.007971 BBM_02g04265.3564 0.00104 0.815476 0.092835 0.893672 BBM_03g02305.2730 0.013936 0.747024 0.640078 1.433619 BBM_03g03046.750 0.000249 0.877976 0.130485 0.911207 BBM_03g01622_e1of3_s1of1.1464 0.005429 0.779762 0.360475 1.138501 BBM_02g00975.2145 0.006667 0.824405 0.01446 0.757339 BBM_02g00810_s2.800 0.00166 0.848214 0.136859 0.905849 BBM_04g08570.3912 0.000561 0.860119 0.024639 0.787656 BBM_03g01705_e1of5_s1of1.4356 0.002535 0.872024 0.233708 0.995128 BBM_04g07915_s1.3376 0.000111 0.883929 0.154629 0.908279 BBM_01g00985.2215 0.002099 0.785714 0.426908 1.176901 BBM_03g00960.4212 0.000121 0.860119 0.078647 0.822714 BBM_03g03925.1879 0.008902 0.803571 0.03924 0.702946 BBM_02g01875.2757 0.007509 0.764881 0.02689 0.76859 BBM_04g05680.1865 0.003561 0.764881 0.031809 0.751091 BBM_04g09295.3890 0.006017 0.758929 0.036413 0.754353 BBM_02g01015_e1of6_s1of1.4036 0.011264 0.791667 0.07767 0.635066 BBM_03g02515_s1.431 0.036854 0.669643 0.472547 1.182825 BBM_03g00315_s2.3982 0.000476 0.815476 0.078946 0.788293 BBM_04g05260_s1.2831 0.00137 0.776786 0.257747 0.966254 BBM_03g02830.468 0.008461 0.839286 0.125011 0.828582 BBM_01g01800.1195 0.005549 0.782738 0.243352 0.942951 BBM_02g04285.2351 0.008494 0.797619 0.245089 0.944029
[0061] IgG and IgM antigens can be ranked according to its relative reactivities. Generally, an antigen with higher mean positive value, lower mean negative value, and lower t-test p-value (and/or higher AUC value) would be, ranked higher as such antigen is predicted to be highly specific to only infected population and show stronger signal (higher antibody titer) in the test. Table 2 illustrates an exemplary group of top candidate antigens that can be used as biomarkers for B. microti infection or onset/progress of Babesiosis. Each antigen in this exemplary group is ranked based on its specificity and sensitivity to IgG and IgM reactivity data.
TABLE-US-00002 TABLE 2 Description Gene.ID ID IgG.Rank IgM.Rank Total Rank BMG09; BMN1-9; BmSA1 BBM_03g00785 BBM_03g00785 1 1 1 rhoptry neck protein 2 BBM_03g04695 BBM_03g04695_s1 2 2.5 2 hypothetical protein BBM_01g00985 BBM_01g00985 3 16.5 3 hypothetical protein BBM_03g00947 BBM_03g00947 4 2.5 4 hypothetical protein BBM_03g00960 BBM_03g00960 5 5 5 rhoptry neck protein 2 BBM_03g04695 BBM_03g04695_s2 6 20.5 6 Nucleoporin FG repeat region BBM_03g02345 BBM_03g02345 7 25.5 7 BMN family (2) BBM_04g09980 BBM_04g09980 8 25.5 8 DNA mismatch repair protein BBM_03g03480 BBM_03g03480 10 35.5 9 hypothetical protein BBM_04g06560 BBM_04g06560 10 31 10 hypothetical protein BBM_02g04285 BBM_02g04285 10 33 11 Actin BBM_03g02390 BBM_03g02390 12 23 12 BMN family (2) BBM_02g04265 BBM_02g04265 14 6 13 hypothetical protein BBM_04g05080 BBM_04g05080 14 38 14 BMN family-2 BBM_03g00020 BBM_03g00020 14 14 15 hypothetical protein BBM_02g02608 BBM_02g02608 16 35.5 16 BMN family (2), MN-10 BBM_02g04280 BBM_02g04280 17 29.5 17 BMG08; BMN family-1 BBM_02g04260 BBM_02g04260 18 29.5 18 S1/P1; Nuclease; PredGPI BBM_02g03140 BBM_02g03140 9.5 19 BMG14 BBM_02g00896 BBM_02g00896 20 24 20 BMG20 BBM_03g03430 BBM_03g03430 21 4 21 BMN family (2) BBM_02g00010 BBM_02g00010 22.5 16.5 22 BMG07, N1-21a orthologue BBM_02g04275 BBM_02g04275 22.5 37 23 BMG10, BMN family, CDS BBM_03g00790 BBM_03g00790 24.5 28 24 hypothetical protein BBM_02g02940 BBM_02g02940 24.5 7 25 hypothetical protein, No PredGBBPMI BBM_04g07556 BBM_04g07556 26 27 26 BMG13, Sexual stage antigen, BBM_04g07810 BBM_04g07810 27 34 27 Protein dopey-1 BBM_02g04060 BBM_02g04060_s2 28 8 28 hypothetical protein BBM_04g06750 BBM_04g06750 29.5 19 29 hypothetical protein BBM_01g01510 BBM_01g01510 29.5 22 30 leucyl aminopeptidase BBM_02g03960 BBM_02g03960 31 39 31 structural maintenance of BBM_01g02740 BBM_01g02740_s2 32.5 11 32 hypothetical protein BBM_02g04250 BBM_02g04250 32.5 32 33 structural maintenance of BBM_01g02740 BBM_01g02740_s1 34 13 34 Vacuolar protein sorting-assoc BBM_04g05345 BBM_04g05345_s2 35 12 35 BMG12, BLAST homologies BBM_03g02840 BBM_03g02840 36 16.5 36 BMN family (1), pseudo gene BBM_02g00005 BBM_02g00005 37 9.5 37 hypothetical protein BBM_01g02415 BBM_01g02415_s1 38 20.5 38 hypothetical protein BBM_03g04760 BBM_03g04760 39 16.5 39
[0062] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms comprises and comprising should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.