Patent Grant
12092640
A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference.
Rickettsia spp., gram-negative obligatly intracellular alphaproteobacteria, are of global medical and veterinary health importance due to their endemicity and re-emergence. From the clinical and antigenic perspective, rickettsial diseases are classified in two groups, spotted fever and typhus. The spotted fever group rickettsiae are transmitted by ticks and include a number of species. The most virulent are R. rickettsii, the agent of Rocky Mountain spotted fever, and R. conorii, the agent of Mediterranean spotted fever (boutonneuse fever), a disease prevalent throughout the Mediterranean, Africa, the Middle East, and India. In humans, the spotted fevers present as acute fever, headache, maculopapular rash, and vascular leakage that can lead to significant morbidity and mortality due to pulmonary and cerebral edema, particularly if there are delays in diagnosis or treatment. There is a need for early diagnosis and administration of treatments as soon as possible.
Infections caused by spotted fever rickettsiae can result in hospitalization, development of complications, and, not infrequently, death in previously healthy individuals of any age. Unfortunately, correct diagnosis still depends on clinical suspicion and a rapid response to empirical treatment with doxycycline. In addition, in tropical areas, there is a long list of febrile syndromes with similar initial clinical presentation. Most of these diseases are of viral etiology (e.g., dengue); thus, the subjects presenting with febrile symptoms are not administered antibiotic treatment.
There remains a need for additional methods and compositions for diagnosing early (differentiating from similar presenting viral infections) and initiating treatment for Rickettsia infections as soon as possible.
One of the major problems with effectively controlling spotted fever rickettsioses is the lack of a specific diagnostic assay to detect the acute stage of infections. The clinical diagnosis of a rickettsial infection relies on the patient's symptoms, history of possible exposure to infected arthropods or travel to an endemic area, and supporting data from laboratory diagnostic assays. However, the current laboratory diagnostic assays for spotted fever rickettsioses are problematic because they are: (1) Too late for physicians to make a specific and prompt treatment plan. At present, serology is the gold standard for diagnosis of spotted fever rickettsioses. However, antibody response often occurs after a time at which treatment is effective; detection of an antibody response is too late. (2) Diagnostics are not reliable. While molecular detection of bacterial DNA and immunohistochemical analysis of rickettsial antigen in the skin biopsy specimens could yield a diagnosis during the acute stage of rickettsial infection, only 40-50% of the patients present with rash or eschar. (3) Diagnostics are not sufficiently sensitive. Because of the endothelial tropism of rickettsiae, molecular genetic approaches often have poor sensitivity in blood samples, except in the late phase of fatal cases. (4) Diagnostics may not be applicable. Laboratory isolation and culture of rickettsiae from clinical samples for diagnosis require technical expertise and specialized facilities, a biosafety level-3 laboratory. Such facilities may not be readily accessible. Thus, early diagnostic assays for spotted fever rickettsioses are urgently needed.
One solution to the above described problems is the development of detection methods and identification of detection targets for sensitive and specific laboratory tests to diagnose and monitor rickettsial infections during the earliest stages of the disease and to assess the degree of illness (host response).
In certain aspects, Rickettsial protein RC0497 (SEQ ID NO:1) can be measured in a sample, e.g., body fluids (plasma, serum, urine), to diagnose rickettsial infection. In specific aspects, tryptic peptides of RC0497 can be detected. The peptides include peptides having, consisting essentially of, or consisting of the amino acid sequence HDLVGLGEVTVNR (SEQ ID NO:2), LLLSLDSTGEK (SEQ ID NO:3), ELAEAGFGR (SEQ ID NO:4), SDFPAEQIGK, (SEQ ID NO:5), EYHNDLTDQAFYAGK (SEQ ID NO:6), EYGYGVESTSTFDQFTQQAVR (SEQ ID NO:7), and/or GASVHYIIDK (SEQ ID NO:8). RC0497 protein or peptide fragments thereof can be measured in body fluids (plasma, serum, urine) alone or in combination for diagnosing rickettsial infection. In certain embodiments the protein and the peptide fragments can be derived from SEQ ID NO:9, SEQ ID NO:10, or a peptide of
In certain aspects, circulating RC0497 or its tryptic peptides can be detected to diagnose R conorii (Mediterranean spotted fever) or R rickettsii (Rocky Mountain spotted fever) infections. In certain aspects the sample is affinity enriched for one or more peptides. Certain embodiments include affinity enrichment-mass spectrometry method (IP-SRM or IP-PRM) for detection of circulating RC0497 or its tryptic peptides in a sample of a subject. In certain aspects, the subject has a rickettsial infection or is suspected of being exposed to rickettsia, or is or has been present in a geographic region where rickettsial infections are prevalent. Rickettsial strains to be detected can include R. conorii, R. rickettsii, R. monacensis, R. amblyommates, R. parkeri, R. sibirica, R. africae, R. phihpii, R. japonica and others (see Table 1).
In certain embodiments a group of host response proteins can be evaluated. The levels of host response proteins can be indicative or used in combination with the rickettsial peptides described herein to determine mild versus severe rickettsial infections (i.e., used as an indicator of prognosis). In certain aspects, a panel of RC0497 and/or fragments thereof and a panel of host response proteins can be used to indicate or assess treatment options and/or treatment response. One embodiment includes a clinical test to diagnose rickettsial infection. One embodiment includes a number of clinical tests over time to monitor the results of treating a rickettsial infection. A panel of rickettsial and host response proteins can be used as an indicator of the response to therapy and to monitor therapeutic efficacy of a treatment.
A “sample” or “biological sample” in terms of the invention means a sample of biological tissue or fluid. Examples of biological samples are sections of tissues, blood, blood fractions, plasma, serum, urine or samples from other peripheral sources. A biological sample may be provided by removing a sample of cells from a subject, but can also be provided by using a previously isolated sample. For example, a tissue sample can be removed from a subject suspected of having a disease by conventional biopsy techniques. In a preferred embodiment, a blood sample is taken from the subject. According to the invention, the biological sample preferably is a blood or a serum sample.
“Polypeptide” refers to any peptide or protein comprising amino acids joined by peptide bonds or modified peptide bonds. “Polypeptide” refers to short chains, including peptides, oligopeptides or oligomers, and to longer chains, including proteins.
“Substantially similar” with respect to amino acid sequences, means at least about 65% identity between two or more sequences. Preferably, the term refers to at least about 70% identity between two or more sequences, more preferably at least about 75% identity, more preferably at least about 80% identity, more preferably at least about 85% identity, more preferably at least about 90% identity, more preferably at least about 91% identity, more preferably at least about 92% identity, more preferably at least about 93% identity, more preferably at least about 94% identity, more preferably at least about 95% identity, more preferably at least about 96% identity, more preferably at least about 97% identity, more preferably at least about 98% identity, and more preferably at least about 99% or greater identity. Such identity can be determined using algorithms known in the art, such as the mBLAST algorithm.
“Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric and polymeric forms of each isotype, unless otherwise specified.
“Functional fragments” of such antibodies comprise portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, functional fragments can comprise at least the CDRs of either the heavy chain or light chain variable region. Functional fragments can also comprise the heavy chain or light chain variable region, or sequences that are substantially similar to the heavy or light chain variable region. Further suitable functional fragments include, without limitation, antibodies with multiple epitope specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as Fab, F(ab′)2, Fd, Fabc, and Fv molecules, single chain (Sc) antibodies (also called ScFv), individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. All antibody isotypes can be used to produce functional fragments of the antibodies herein. Functional fragments can be recombinantly or synthetically produced, with natural or unnatural nucleic acid or amino acid molecules.
The antibodies or functional fragments thereof of the disclosed subject matter can be generated from any species. The antibodies or functional fragments thereof described herein can be labeled or otherwise conjugated to various chemical or biomolecule moieties, for example, for therapeutic or diagnostic or detection or treatment applications. The moieties can be cytotoxic, for example, bacterial toxins, viral toxins, radioisotopes, and the like. The moieties can be detectable labels, for example, fluorescent labels, radiolabels, biotin, and the like, which are known in the art.
Antibodies of the present invention can be used to detect the various proteins and peptide described, the antibodies can be labeled with a detectable moiety, such as enzymes or fluorophores. There are a wide variety of fluorophore labels that can usefully be attached to the antibodies of the present invention. Common useful fluorophores can be fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Texas Red, Cy3, Cy5, fluorescence resonance energy tandem fluorophores such as PerCPCy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7. Other fluorophores include, inter alia, Alexa Fluor® 350, Alexa Fluor® 488, Alexa 25 Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, OR, USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR, USA), and Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, all of which are also useful for fluorescently labeling the antibodies of the present invention. For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the present invention can usefully be labeled with biotin. When the antibodies of the present invention are used, e.g., for western blotting applications, they can usefully be labeled with radioisotopes, such as 33P, 32P, 35S, 3H, and 125I.
An “anti-RC0497 antibody” described herein detects or binds an epitope of RC0497 protein. An anti-RC0497 antibody can comprise a light chain variable region and a heavy chain variable region.
The antibodies or functional fragments thereof described herein have binding affinities (in M) for RC0497 or peptides thereof that include a dissociation constant (KD) of less than 1×10−2. In some embodiments, the KD is less than 1×10−3. In other embodiments, the KD is less than 1×10−4. In some embodiments, the KD is less than 1×10−5. In still other embodiments, the KD is less than 1×10−6. In other embodiments, the KD is less than 1×10−7. In other embodiments, the KD is less than 1×10−8.
The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of a rickettsial infection, including any objective or subjective parameter such as abatement, diminishing of symptoms such as fever, pulmonary or cerebral edema. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and/or psychiatric evaluations.
As used herein, the term “subject” refers to any mammal, including both human and other mammals. Preferably, the methods of the present invention are applied to human subjects.
As used herein, the term “antigen” is a molecule capable of being bound by an antibody. The structural aspect of an antigen, e.g., three-dimensional conformation or modification, is referred to herein as an “antigenic determinant” or “epitope.” Thus, antigenic determinants or epitopes are those parts of an antigen that are recognized by antibodies. An antigenic determinant need not be a contiguous sequence or segment of protein and may include various sequences that are not immediately adjacent to one another, i.e., a conformational epitope.
The term “antibody” or “immunoglobulin” is used to include intact antibodies and binding fragments/segments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen. Fragments include separate heavy chains, light chains, Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments/segments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. The term “antibody” also includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai and Lachmann, Clin Exp Immunol 79:315-21, 1990; Kostelny et al., J. Immunol. 148:1547-53, 1992.
The term “isolated” can refer to a polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques) of their source of origin, or chemical precursors or other chemicals (when chemically synthesized).
Moieties of the invention, such as polypeptides, peptides, antigens, or immunogens, may be conjugated or linked covalently or noncovalently to other moieties such as adjuvants, proteins, peptides, supports, fluorescence moieties, or labels. The term “conjugate” or “immunoconjugate” is broadly used to define the operative association of one moiety with another agent and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical “conjugation.”
“Prognosis” refers to a prediction of how a patient will progress, and whether there is a chance of recovery. “Rickettsiosis prognosis” generally refers to a forecast or prediction of the probable course or outcome of a rickettsial infection. As used herein, prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient—and/or response rate in a group of patients susceptible to or diagnosed with a rickettsiosis. Prognosis also includes prediction of favorable responses to treatments, such as a conventional antibiotic therapy.
In one embodiment, the marker level is compared to a reference level representing the same marker. A reference level may be a reference level of from a different subject or group of subjects know to have or not having rickettsial infection. The reference level may be a single value or may be a range of values. The reference level can be determined using any method known to those of ordinary skill in the art. In some embodiments, the reference level is an average level of expression determined from a cohort of subjects serving as negative or positive controls/reference. The reference level may comprise data obtained at the same time (e.g., in the same hybridization experiment) as the patient's individual data, or may be a stored value or set of values, e.g., stored on a computer, or on computer-readable media. If the latter is used, new patient data for the selected marker(s), obtained from initial or follow-up samples, can be compared to the stored data for the same marker(s) without the need for additional control experiments.
The phrase “specifically binds” or “specifically immunoreactive” to a target refers to a binding reaction that is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologics. Thus, under designated immunoassay conditions, a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologics present in the sample. Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein/peptide. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and/or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and/or method.
As used herein, the transitional phrases “consists of” and “consisting of” exclude any element, step, or component not specified. For example, “consists of” or “consisting of” used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of” or “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of” or “consisting of” limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.
As used herein, the transitional phrases “consists essentially of” and “consisting essentially of” are used to define a chemical composition and/or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.
The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment, and not intended to intimate that the scope of the disclosure, including the claims, is limited to that embodiment.
The clinical manifestations of acute MSF are a consequence of the tropism of rickettsiae for endothelial cells. Rickettsial organisms bind Ku70 on cholesterol-rich microdomains and enter endothelial cells through a calcium-dependent zipper-like entry mechanism. Viable organisms subsequently lyse intracellular phagosomes via phospholipase D and hemolysin activities, and replicate in the cytoplasm. A characteristic of the spotted fever group is that R. conorii organisms spread intercellularly as a consequence of directional actin polymerization without producing early cellular injury. Oxidative stress produced by replication of R. conorii results in peroxidation of endothelial membranes and disruption of the endothelial barrier function producing vascular leakage.
The pulmonary and cerebral edema in patients infected with rickettsiae can lead to significant morbidity and mortality if there are delays in diagnosis and treatment. The reference standard for diagnosis of rickettsial diseases is a fourfold rise in antibody titer by the indirect immunofluorescence assay (IFA) assay using paired serum samples obtained soon after illness and 2-4 weeks later. Patients usually do not have diagnostic serum antibody titers during the first week of illness, and a negative result by IFA does not exclude the diagnosis. Therefore, many cases are initially misdiagnosed accounting for adverse outcomes, and consequently, the epidemiology of the disease is under-reported. A highly specific and sensitive biomarker for early diagnosis of rickettsial infection is greatly needed.
The RC0479 protein has been found in both in vitro cell culture and in vivo animal models of spotted fever rickettsial infections by using an affinity enrichment-mass spectrometry method (IP-SRM or IP-PRM). RC0497 is a diagnostic biomarker for spotted fever rickettsioses. Embodiments are directed to diagnostic assays developed by detecting this biomarker. These assays are expected to provide specific diagnostic information at the early stage of spotted fever rickettsioses when treatment can make a difference in patient management.
The protein RC0497 (SEQ ID NO:1) was detected in high concentrations in cell culture supernatant and with high confidence (
Rickettsial proteins were identified as diagnostic in vivo. Plasma samples from four animal groups (17 samples in total) Group 1: uninfected (n=5 mice); Group 2: sub-lethal dose (n=6 mice); Group 3: lethal dose (n=3 mice); and Group 4 dead (n=3 mice). The plasma samples were subjected to four analyses: (1) Global protein profiling without prefraction of plasma proteins or peptides; (2) Targeted profiling for rickettsia protein RC0497 using selected reaction monitoring (SRM); (3) targeted profiling for RC0497 using parallel reaction monitoring (PRM); and (4) strong cation exchange (SCX) chromatography-LC-MS/MS analysis.
The first attempt of global protein profiling without using any forms of prefraction technology primarily identified a panel of host response proteins that associate with disease severity. RC0497 was not identified in this experiment primarily due to the interference from high-abundance host proteins in mass spectrometry analysis. Two proteotypic peptides of RC0497, LLLSLDSTGEK (SEQ ID NO:3) and ELAEAGFGR (SEQ ID NO:4), were identified in sublethally and lethally infected mice by PRM. Shown in
RC0497 is conserved in spotted fever strains. Analysis indicates that RC0497 and the diagnostic tryptic peptides are highly conserved across rickettsial strains (
Proteotypic peptides are conserved in the Rickettsia genus. Conservation of the proteotypic peptides for RC0497 were analyzed in regard to other known Rickettsia species. In addition to R. conorii, and R. rickettsii, the diagnostic peptides are found in R. conorii, R. rickettsii, R. monacensis, R. amblyommates, R. parkeri, R. sibirica, R. africae, R. phihpii, R. japonica and others These data indicate that the IP-SRM and IP-PRM assays will be diagnostic for a wide variety of rickettsial infections.
Host response proteins. The understanding of the host response to rickettsial infection has been advanced by the development of a standardized model of endothelial cell infection using primary human umbilical vein cells (HUVECs). Proteomics were applied for measurement and identification of rickettsial proteins in the golgi, plasma membrane, or secreted proteins. The proteins selected are based on the identification of specific protein fragments in infected human endothelial cells, which could not be predicted based on available genomic sequence. Similarly, the host response proteins are combinations of proteins that represent the endothelial response to infection.
Identification of host response proteins were extended to those associated with sublethal vs lethal infections. Distinct protein profiles were obtained for each disease type as shown in the heat map in
Identification of protein panel that differentiates sublethal disease (SLD) from control. The differentially expressed proteins were subjected to statistical analysis for microarray (SAM) to identify those proteins that were significantly different by outcome. Over 140 proteins were significant; of these the top 5 informative markers are shown in Table 2 below. The protein distribution is shown in
R. buchneri
R.
monacensis
Candidatus
Rickettsia
amblyommii
R.
rhipicephali
Candidatus
Rickettsia
amblyommii
Rickettsia
endosymbiont
pacificus
Rickettsia
argasii
Rickettsia
rickettsii
Rickettsia
canadensis
Rickettsia
massiliae
Rickettsia
rickettsii
Rickettsia
africae
Rickettsia
endosymbiont
scapularis
Rickettsia
heilongjiangensis
Rickettsia
japonica
Rickettsia
philipii
Rickettsia
massiliae
Rickettsia
amblyommii
Rickettsia
montanensis
Rickettsia
rhipicephali
Identification of protein panel that differentiates lethal disease (LD) vs SLD. The top 5 informative markers are shown in Table 2. The abundance of the proteins are plotted in
A panel of polyclonal and monoclonal antibodies directed against RC0497 have been developed. More importantly, these antibodies are able to specifically bind RC0497 by both immunoblotting and ELISA. Recombinant RC0497 was plated as the antigen at the concentration of 0.07 μg/μl. One of the monoclonal antibody clones (7F) was used at different concentrations to detect RC0497. No significant signal was detected in the negative controls. These results demonstrate the feasibility of detecting the diagnostic biomarker RC0497 using immunoassay.
Certain aspects are directed to a sandwich ELISA to detect RC0497 in the serum of patients at the acute stage of spotted fever rickettsioses using a time- and cost-efficient strategy. In brief, the optimal mouse monoclonal antibody against RC0497 will be used as the capture antibody after coating the plate. After blocking, patient serum, which contains RC0497 or its fragments, will be added to bind to the capture antibody. Purified rabbit polyclonal antibody against RC0497 will be biotinylated and used as the detection antibody. The antigen, RC0497, will be bound between these two layers of antibodies. The ultraAvidin-HRP conjugate will be added prior to TMB substrate solution. The enzyme-substrate reaction can be used as the detection signal. The capture and detection antibodies must be chosen to prevent cross-reactivity or competition of binding sites. The specificity and sensitivity can be measured using serum samples from healthy individuals and infected patients.
In an aspect, the invention provides a method of detecting one or more biomarker (e.g., RC0497 or a fragment thereof) in a biological sample comprising: (a) contacting a biological sample with a reagent designed to determine a presence or level of the one or more biomarker, wherein the one or more biomarker is selected from the RC0497 or a fragment thereof; and (b) identifying the one or more biomarkers in the biological sample, thereby detecting the one or more biomarker in the biological sample.
Biomarkers. A biomarker, such as the RC0497 protein or fragments thereof, is a biomolecule that is differentially present in a sample taken from a subject of one phenotypic status (e.g., having a disease) as compared with another phenotypic status (e.g., not having the disease). A biomarker is differentially present between different phenotypic statuses if the mean or median expression level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. As such, they are useful as markers for disease (diagnostics), therapeutic effectiveness of a drug (theranostics) and of drug toxicity.
The current invention seeks to develop method for identifying patients having rickettsia infection based on multiple factors including clinical features, biochemical assays, and expression profiling.
Assays. In certain aspects, the biomarkers of this invention can be measured or detected by immunoassay. Immunoassay requires biospecific capture reagents, such as antibodies, to capture the biomarkers. Antibodies can be produced by methods well known in the art, e.g., by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies.
Embodiments described herein contemplate traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays. In the SELDI-based immunoassay, a biospecific capture reagent for the biomarker is attached to the surface of an MS probe, such as a pre-activated ProteinChip array. The biomarker is then specifically captured on the biochip through this reagent, and the captured biomarker is detected by mass spectrometry.
Kits. In another aspect, the present invention provides kits for qualifying rickettsia status, which kits are used to detect biomarkers described herein. In one embodiment, the kit comprises a solid support, such as a chip, a microtiter plate or a bead or resin having a capture reagent attached thereon, wherein the capture reagent binds a biomarker of the invention. Thus, for example, the kits of the present invention can comprise mass spectrometry probes for SELDI, such as ProteinChip® arrays. In the case of biospecific capture reagents, the kit can comprise a solid support with a reactive surface, and a container comprising the biospecific capture reagent.
The kit can also comprise a washing solution or instructions for making a washing solution, in which the combination of the capture reagent and the washing solution allows capture of the biomarker or biomarkers on the solid support for subsequent detection by, e.g., mass spectrometry. The kit may include more than type of adsorbent, each present on a different solid support.
In a further embodiment, such a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. For example, the instructions may inform a consumer about how to collect the sample, how to wash the probe or the particular biomarkers to be detected.
In yet another embodiment, the kit can comprise one or more containers with biomarker samples, to be used as standard(s) for calibration.
Examples. The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
A major challenge in the development of rickettsial diagnostics is that the infection is localized to the vascular endothelium, and bacteremia is not typically present. To circumvent this limitation, markers for early R. conorii infection are identified (
A. Materials and Methods
Reagents. All reagents and solvents in LC-MS/MS analyses were ACS grade. Ammonium bicarbonate (ABC), 2,2,2,-trifluoroethanol (TFE), and acetic acid were purchased from Sigma-Aldrich. Iodoacetamide (IDA), dithiothreitol (DTT), acetonitrile (ACN), formic acid, and methanol were purchased from Thermo Scientific (Waltham, MA, USA). Urea ultra was from MP Biomedicals (Santa Ana, CA, USA). Sequencing-grade modified trypsin and LysC were from Promega (Madison, WI, USA). Recombinant rickettsial putative N-acetylmuramoyl-L-alanine amidase RC0497 was purchased from MyBiosource (San Diego, CA).
Rickettsia. R. conorii (Israeli spotted fever strain, ISF) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). For in vitro cell infection, R. conorii ISF strain was cultivated in Vero cells. To purify rickettsiae, cells were homogenized, and rickettsiae were diluted in a 10% suspension of sucrose-phosphate-glutamate (SPG) buffer (0.218 mM sucrose, 3.8 mM KH2PO4, 7.2 mM K2HPO4, 4.9 mM mono-sodium glutamic acid, pH 7.0) and stored at −80° C. For mouse inoculation, R. conorii (Malish 7 strain) was obtained from the American Type Culture Collection (ATCC; Manassas, Va.; catalog no. VR-613). Rickettsiae were propagated in the yolk sacs of specific-pathogen-free embryonated chicken eggs. The concentrations of stock rickettsiae cultured in both yolk sacs and cell culture were determined by plaque assay. The rickettsial stock was stored at −80° C. until used, and all the experiments described in this study were performed in a certified biosafety level 3 (BSL3) laboratory at the University of Texas Medical Branch at Galveston (UTMB).
Ethical approval. Animal experiments were performed according to the NIH Guide for Care and Use of Experimental Animals and approved by the University of Texas Medical Branch (UTMB) Animal Care and Use Committee (IACUC No. 90-07082). This study of human subjects was conducted under approval from the UTMB Institutional Review Board (No. 08-258), and was compliant with all applicable federal regulations governing the protection of human subjects.
Cell Cultures. Pools of HUVECs were established from individual human umbilical cords grown in supplemented EGM-Plus endothelial cell growth medium (Lonza, catalog no. CC-5035) without the addition of GA-1000 (gentamicin sulfate and amphotericin-B) or fetal bovine serum. The cells were subcultured when the monolayer became confluent. In this study, the cells were used between passages 3 and 4. For infection, 15×106 primary HUVECs in T175 flasks were infected in BSL-3 containment, and subsequently the conditioned medium was collected 24 h post infection. The conditioned medium was filtered through a 0.22 μm membrane filter to remove infectious rickettsiae in accordance with approved protocols.
Trypsin digestion of the secretome of HUVECs infected with R. conorii. The isolation of secretome was performed. Briefly, the conditioned medium was collected and centrifuged at 2000×g at 4° C. for 20 min to remove any dead cells. The supernatant was centrifuged at 10,000×g at 4° C. for 10 min to remove cell debris. The supernatant was further concentrated using Amicon ultra-4 centrifugal filters-3K (Millipore, Billerica, MA, USA). Then 200 μL of 8 M urea was added into centrifugal filters to denature the proteins. Next, the proteins were reduced with 10 mM dithiothreitol (DTT) for 30 min, followed by alkylation with 30 mM iodoacetamide for 60 min at room temperature in the dark. The centrifugal filters were centrifuged at 14,000×g at 4° C. for 15 min, and 200 μL of 50 mM of ammonium bicarbonate (pH 8.0) was added into the sample. The centrifugal filters were centrifuged at 14,000×g at 4° C. for 15 min again. The sample that remained in the filter was transferred into a 0.5-mL microcentrifugation tube. The proteins were digested with 1.0 μg LysC-trypsin (Promega) for 12 h at 37° C., then diluted, and further digested with 1.0 μg trypsin (Promega) for 16 h at 37° C. The digestion was terminated with 0.5% trifluoroacetic acid. The peptides were desalted on a reversed-phase SepPak C18 cartridge (Waters), and eluted with 80% acetonitrile. The eluate was dried in a SpeedVac and the peptides were resuspended in 2% acetonitrile-0.1% trifluoroacetic acid for LC-MS/MS analysis.
Label-free quantification of protein expression. The desalted peptides were reconstituted in 30 μ4% ACN/0.1% formic acid. All peptide samples were separated on an online nanoflow Easy nLC1000 UHPLC system (Thermo Scientific) and analyzed on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA). 10 μl of sample was injected onto a capillary peptide trap column (Acclaim® Pepmap 100, 75 μm×2 cm, C18, 3 μm, 100 Å, Thermo Scientific). After sample injection, the peptides were separated on a 25-cm UHPLC reversed phase column (Acclaim® Pepmap 100, 75 μm×25 cm, C18, 2 μm, 100 Å, Thermo Scientific) at a flowrate of 300 nL/min. A 2-h linear gradient from 2% solvent A (0.1% formic acid in water) to 35% solvent B (0.1% formic acid in acetonitrile) was used for each LC-MS/MS run. Data-dependent acquisition was performed using the Xcalibur 2.3 software in positive ion mode at a spray voltage of 2.1 kV. Survey spectra were acquired in the Orbitrap with a resolution of 70,000, the maximum injection time of 80 ms, an automatic gain control (AGC) of 1×106, and a mass range from 400 to 1400 m/z. The top 15 ions in each survey scan were selected for higher-energy collisional dissociation (HCD) scans with a resolution of 17,500. For all higher-energy collisional dissociation (HCD) scans, collision energy was set to 30, the maximum inject time was 60 ms and the AGC was 1×105. Ions selected for MS/MS were dynamically excluded for 30 s after fragmentation.
All data were analyzed with the MaxQuant software (version 1.5.2.8) 20, 21 with the Andromeda search engine. The false discovery rate (FDR) was set to 1% for both proteins and peptides, and specified a minimum length of 7 amino acids. The Andromeda search engine was used for the MS/MS spectra search against a combined SwissProt human and rickettsial database (downloaded on December 2015 containing 20,193 human protein entries, 4,476 Rickettsial protein entries and 247 contaminants). Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and a maximum of two missed cleavages. Carbamidomethylation of cysteine was selected as fixed modification and methionine oxidation as variable modifications. The ‘match between runs’ feature of MaxQuant was used to transfer identifications to other LC-MS/MS runs based on their masses and retention time (maximum deviation 0.7 min), and this was also used in quantification experiments. Quantifications were performed with the label-free algorithms in Maxquant. At least one ‘razor peptide’ was required for quantification. The Perseus platform was used to analyze the Maxquant output, including statistics, Hierarchical clustering, and principal component analysis (PCA). Reversed identifications and proteins identified only by site modification were strictly excluded from further analysis. After filtering (2 valid values in at least one group), remaining missing values were imputed from a normal distribution (width: 0.3 of standard deviation; down shift: 1.8 of standard deviation). Student's t-test was performed to identify the significantly differentially expressed proteins with a Permutation-based FDR<0.01. The unsupervised hierarchical clustering and heat map were based on protein expression. The rows of the heat map indicate the proteins, and the columns indicate the samples. The log 2 ratios of each protein were z-score normalized for each row. Hierarchical clustering of the z-normalized log 2 ratio was performed using Euclidean distances between means. The number of clusters was set as 300. Genome ontology enrichment analysis of molecular functions and biological function in differentially expressed proteins used Panther (URL pantherdb.org/). This classification uses an evolutionary framework to infer protein functions in a species-independent manner.
Stable Isotope Dilution (SID)-Selected Reaction Monitoring (SRM)-MS validation of differentially expressed secreted proteins. The SID-SRM-MS assays of selected proteins were developed for each targeted protein, two or three peptides were initially selected, and then the sensitivity and selectivity of these were experimentally evaluated. The peptide with best sensitivity and selectivity was selected as the surrogate for that protein. For each peptide, 3-5 SRM transitions were monitored. The peptides were chemically synthesized incorporating isotopically labeled [13C615N4] arginine or [13C615N2] lysine to a 99% isotopic enrichment (Thermo Scientific, San Jose, CA). The amount of stable isotope labeled standard (SIS) peptides was determined by amino acid analysis. The proteins were trypsin digested on the beads as described above. The tryptic digests were then reconstituted in 30 μl of 5% formic acid-0.01% TFA. An aliquot of 10 μl of 50 fmol/μL diluted SIS peptides was added to each tryptic digest. These samples were desalted with a ZipTip C18 cartridge. The peptides were eluted with 80% ACN and dried. The peptides were reconstituted in 30 μl of 5% formic acid-0.01% TFA and were directly analyzed by liquid chromatography (LC)-SRM-MS. LC-SRM-MS analysis was performed with a TSQ Vantage triple quadrupole mass spectrometer equipped with a nanospray source (Thermo Scientific, San Jose, CA). About 8-10 targeted proteins were analyzed in a single LC-SRM run. The online chromatography was performed using an Eksigent NanoLC-2D HPLC system (AB SCIEX, Dublin, CA). An aliquot of 10 μL of each of the tryptic digests was injected on a C18 reverse-phase nano-HPLC column (PicoFrit™, 75 μm×10 cm; tip ID 15 μm) at a flow rate of 500 nL/min with a 20-min 98% A, followed by a 15-min linear gradient from 2-30% mobile phase B (0.1% formic acid-90% acetonitrile) in mobile phase A (0.1% formic acid). The TSQ Vantage was operated in high-resolution SRM mode with Q1 and Q3 set to 0.2 and 0.7-Da Full-Width Half Maximum (FWHM). All acquisition methods used the following parameters: 2100 V ion spray voltage, a 275° C. ion transferring tube temperature, a collision-activated dissociation pressure at 1.5 mTorr, and the S-lens voltage used the values in S-lens table generated during MS calibration.
All SRM data were manually inspected to ensure peak detection and accurate integration. The chromatographic retention time and the relative product ion intensities of the analyte peptides were compared to those of the stable isotope labeled standard (SIS) peptides. The variation of the retention time between the analyte peptides and their SIS counterparts should be within 0.05 min, and the difference in the relative product ion intensities of the analyte peptides and SIS peptides was below 20%. The peak areas in the extract ion chromatography of the native and SIS version of each signature peptide were integrated using Xcalibur® 2.1. The default values for noise percentage and base-line subtraction window were used. The ratio between the peak area of native and SIS version of each peptide was calculated.
In vivo study. Age- and sex-matched C3H/HeN mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were inoculated intravenously (i.v.) through the tail vein with R. conorii Malish 7 strain with either a sub-lethal dose (SLD) 0.1 LD50 (5×103 PFU) or a lethal dose (LD) 3LD50 (1.5×105 PFU) of ricketssiae. Negative controls were inoculated with 300 μl of SPG buffer alone. Mice were monitored daily for signs of illness including ruffled fur, hunched posture, and decreased activity. Mice were sacrificed on day 4 p.i. Mice were anesthetized by inhalational isoflurane (Isoflurane® USP, Piramal Healthcare Limited, 502321 Andhra Pradesh, India) and euthanized by CO2 inhalation followed by cervical dislocation. Whole mouse blood was recovered by cardiac puncture immediately after euthanasia. All animal experiments were conducted in a certified animal biosafety level 3 (ABSL3) laboratory. Experiments in mice were performed according to the guidelines of the Guide for the Care and Use of Laboratory Animals.
Trypsin digestion of mouse plasma or human serum and label free quantification. Ten microliters of plasma were aliquoted from each sample, and 50 μl of 9 M urea was added. The protein concentration was measured by bicinchoninic acid assay. About 200 μg of proteins from each sample were used for protein digestion. The proteins were first reduced with 10 mM DTT at room temperature for 30 min, followed by alkylation with 30 mM iodoacetamine at room temperature for 1 h. Then, the sample was diluted 2× with 50 mM ammonium bicarbonate (pH 8.0). An aliquot of Lys-C/Trypsin solution (Promega, Madison, WI) was added into each sample at a 100:1 protein:enzyme ratio. The samples were incubated at 37° C. overnight, and the solutions were further diluted 5× with 50 μM of ammonium bicarbonate. An aliquot of Trypsin solution (Promega, Madison, WI) was added into each sample at a 50:1 protein:enzyme ratio. The samples were incubated at 37° C. for 16 h. 10 μL of 10% trifluoroacetic acid was added into each sample to stop the trypsin digestion. Tryptic peptides were desalted on reversed phase tC18 SepPak columns (Waters, Milford, MA) and evaporated to dryness in a vacuum concentrator. The peptides were resuspended in 2% acetonitrile-0.1% trifluoroacetic acid and analyzed by LC-MS/MS as described above. The mass spectrometry data were analyzed with MaxQuant software as described above.
Parallel reaction monitoring (PRM) analysis of Rickettsia protein RC0497, An aliquot of stable isotope labeled peptide (SIS) of rickettsial protein RC0497 (LLLSLDSTGEK[13C615N2] was added into each sample. For PRM analyses, the acquisition employed an orbitrap resolution of 70,000 (@m/z 200), a target AGC value of 3×106, and maximum fill times of 200 ms for full scan; 17,500 (@m/z 200), a target AGC value of 2×105, and maximum fill times of 100 ms for MS2 scan. PRM targeted the pair of peptides of rickettsial protein RC0497 [(native and stable isotope labeled standard (SIS) peptides). All peptide samples were separated on an online nanoflow Easy nLC1000 UHPLC system (Thermo Scientific) and analyzed on an Q Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) as described above. The assessment of the detection of peptides was performed post-acquisition using Skyline version 3.6.0.9321. For each peptide under evaluation, the signals of the five most intense fragment ions (as defined in spectra of SIS peptides of RC0497) were extracted from each corresponding MS/MS spectrum. The MS/MS spectra with at least five fragment ions detected were submitted to spectral matching. The comparison of the relative intensities of these fragments with those defined in the reference composite MS/MS spectrum was performed based on dotp value. In addition, the retention time of the native and SIS peptides was used as an additional acceptance criterion. The variation of the retention time between the analyte peptides and their SIS counterparts should be within 0.05 min.
Generation of anti-RC0497 antibodies. Recombinant protein Rc0497 was expressed in E. coli. After expression, the protein was purified using Ni-resin and dialyzed to remove imidazole and benzamidine to optimize thrombin activity followed by removal of His-tag. Rabbit polyclonal antibodies against RC0497 were generated by Rockland Immunochemicals, Inc. (Limerick, PA). The antibodies then underwent either protein A or affinity purification. The reactivity of polyclonal antibody with RC0497 was confirmed by immunoblotting and immunoprecipitation-SRM.
Immunoprecipitation (IP) of RC0497 and trypsin digestion. About 100 μL of serum was suspended in 1 mL of low ionic strength immunoprecipitation buffer (50 mM NaCl, 25 mM HEPES pH 7.4, 1% IGEPAL CA-630, 10% glycerol, 1 mM fresh DTT, and protease inhibitor cocktail). A aliquot of 4 μg of anti-RC0497 antibody or control IgG (Rabbit polyclonal, Santa Cruz) was used in each IP. The mixture was incubated overnight at 4° C.; then, 30 μL of protein A magnetic beads (Dynabeads, Invitrogen) were added. After incubation at 4° C. for 4 h, the beads were separated from the supernatant with a magnetic stand. The beads were washed with PBS five times before trypsin digestion. The trypsin digestion was performed. The beads were suspended with gentle vortexing for 1 h. The proteins on the beads were reduced with 10 mM DTT for 30 min, then alkylated with 20 mM IDA for 1 h in the dark. An aliquot of 4 μg of sequencing-grade trypsin was added to each sample before a 4 h incubation at 37° C. with gentle shaking; the supernatant was then collected. Another 4 μg of trypsin was then added to the beads, and the sample was incubated at 37° C. overnight with gentle shaking; the supernatant was then collected. After trypsin digestion, the beads were washed twice with 50 μL of 50% ACN, and the supernatants were collected. All of the supernatants were combined and dried with a SpeedVac.
B. Results
Quantitative proteomics analysis of secretome of HUVECs infected with R. conorii. HUVECs are a standardized model for early responses to rickettsial vascular infection. To understand the spectrum of proteins secreted/released by endothelial cells in response to R. conorii infection, a quantitative proteomics analysis of the proteins in the conditioned medium of HUVECs with and without R. conorii infection were conducted (
R. conorii
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
H. sapiens
The changes in the abundance of these proteins in the secretome of HUVECs were further validated with independent quantitative stable isotope dilution (SID)-selected reaction monitoring (SRM)-MS, a “targeted” MS approach for the detection and accurate quantification of proteins in a complex background. SID-SRM-MS provides structural specificity and, therefore, is the most accurate approach available for direct quantification of target proteins in a complex mixture. SRM assays were developed for the measurement of five proteins—RC0497, CSF3, CXCL1, CXCL3, and SQSTM1. Using two proteotypic peptides (SDFPAEQIGK (SEQ ID NO:5) and LLLSLDSTGEK (SEQ ID NO:3)), a marked increase of RC0497 abundance was observed in the conditioned medium of R. conorii-infected HUVECs (
Host responses to R. conorii infection. The genome ontology (GO) biological process enrichment analysis of the 45 up-regulated human proteins found that these proteins are involved in positive regulation of neutrophil chemotaxis, chemokine-mediated signaling pathways, and inflammation response (
Quantitative proteomics analysis of the plasma from mice infected with R. conorii. To identify proteins induced in a small animal model of rickettsial infection, mice were infected with a sub-lethal dose (SLD, n=6) or lethal dose (LD, n=3) of R. conorii; uninfected animals were used as control (Ctrl, n=5). Label-free LC-MS/MS analysis of the mouse plasma identified 608 proteins with 1% FDR. Of these, 534 proteins were quantified in all the samples (Table 4). Comparing the intensity of each protein in biological and technical replicates shows an excellent agreement (Pearson correlation r=0.90-0.96) (
Rickettsia conorii infection. Three experimental groups: sub-lethal dose (SLD, n = 6), lethal-dose
Unsupervised hierarchical clustering of these 397 proteins identified three clusters (
A principal component analysis (PCA) of the 397 significantly changed proteins yielded quantitative separation among the animals in the SLD, LD, and uninfected control groups (
Several protein markers were selected including Saa1/2/3, Lcn2, Ckm, Albg, and Mup8 for further validation with quantitative SID-SRM-MS assays. As shown in
SID-parallel reaction monitoring (PRM)-MS analysis of rickettsia RC0497 protein in mouse plasma. In the analysis of the plasma proteome of the animals infected with R. conorii, no rickettsial protein was identified, including RC0497, probably due to their low-abundance in the mouse plasma. To increase the sensitivity of detection, a targeted SID-PRM assay was developed for RC0497. The stable isotope labeled RC0497 signature peptide LLLSLDSTGE[K(13C6,15N2)] of RC0497 was used to determine the best PRM transitions for this peptide (
Conservation of RC0497 in major spotted fever group rickettsiae. In this study, RC0497 was identified in the conditioned medium of HUVECs infected with R. conorii. To determine whether this protein was conserved in other rickettsial strains, a protein sequence alignment analysis was conducted. It was found that protein RC0497 is highly conserved across diverse rickettsial strains including R. rickettsii, the causative agent of the Rocky Mountain spotted fever (RMSF) (
Verification of RC0497 and host proteins in the serum from patients with acute rickettsiosis. Verification of the presence of RC0497 was attempted in the serum of patients with rickettsial infection with targeted MS analysis, but it was found that the level of RC0497 in the serum of patients with rickettsial infection is much lower than the in vivo model, and the sensitivity of PRM or SRM were not enough for detecting RC0497 in patients' serum. To increase the sensitivity of the assay, an immunoprecipitation (IP)-SRM assay was developed in which the RC0497 protein was captured from serum with an RC0497 specific antibody and followed by SRM analysis of the enriched protein. Because there is no commercial RC0497 antibody, a polyclonal antibody was developed that is directed against RC0497. The reactivity and specificity of the antibody against RC0497 were first examined using immunoblotting. The culture medium and cell lysate of HUVECs infected with R. conorii were collected. The presence of RC0497 in the medium and cell lysate was probed with the anti-RC0497 antibody. The culture medium and cell lysate collected from uninfected cells were used as control. As shown in
Next, 13 de-identified paired serum samples (acute and convalescent) from confirmed cases of Mediterranean spotted fever collected from discarded diagnostic material were used. Serum samples from six healthy individuals free of rickettsial infection were used as control. The diagnosis of MSF infection was made with IFA. IP-SRM-MS assay described above was used to measure the abundance of RC0497 in these samples. As shown in
Also, SRM assays were developed for human host proteins SAA1 and SAA2. Both proteins were significantly up-regulated in the serum of mice infected with R. conorii. The level of these two proteins was measured in the paired human serum collected during the acute phase and convalescence of R. conorii infection. As shown in
The present application claims priority to U.S. Provisional Application No. 62/892,048 filed Aug. 27, 2019 which is incorporated herein by reference.
This invention was made with government support under TR001439 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20030003508 | Raoult | Jan 2003 | A1 |
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| Number | Date | Country | |
|---|---|---|---|
| 20210063394 A1 | Mar 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62892048 | Aug 2019 | US |
