WO 2008/133705
`
`PCT/US2007/082096
`
`
`
`ASSA YS AND DEVICES FOR IDENTIFYING PA THOGENS
`
`BACKGROUND OF THE INVENTION
`
`[0001] Detection of the bacteria that have infected a subject, including metabolites, nucleic acids,
`
`and proteins thereof, is a fundamental component in the diagnosis and treatment of medical
`
`disorders, as well as in research. A number of methodologies are currently in use for detection.
`
`These methodologies can generally be divided into antibody—based diagnostic assays for proteins,
`
`either components of the bacteria or byproducts of the disease, and diagnostic assays for nucleic
`
`acids, such as the genetic material encoding a component of the bacteria.
`
`[0002] Existing methodologies for nucleic acid detection require a high degree of technical
`
`competence for reliability due to the complexity of the reaction conditions (for example, PCR
`
`requires thermocycling) and may be extremely sensitive to contamination resulting in false
`
`positives; they are difficult to use quantitatively rather than qualitatively and thus their sensitivity is
`
`compromised. Further, they often take hours to complete. Methodologies for protein detection
`
`generally rely on conjugation of an enzyme, usually to additional components of the assay, to
`
`increase signal generation and amplification. The use of these additional ligands increases the noise
`
`of the system, with higher background and false positives, and necessitates several levels of control
`
`reactions.
`
`SUMMARY OF THE INVENTION
`
`[0003] Provided are methods of using ribosomes from pathogens to identify the pathogens, e.g., for
`
`diagnostic or treatment purposes. Ribosomes are specific to a particular pathogen, and thus the
`
`identity or presence of a pathogen in a sample may be determined based on determining whether a
`
`polypeptide is able to be produced using a nucleic acid template With ribosomes isolated using a
`
`binding agent specific for the pathogen’s ribosomes. The methods may be performed at a single
`
`temperature, avoiding complex reaction conditions. Further, the amplification of the template by
`
`the ribosomes is rapid — at least about 20,000 target molecules are produced every 10 seconds - and
`
`is very sensitive. Still further, the amplification reaction itself is generic, With only the nucleic acid
`
`template and binding agent used to immobilize the ribosomes differing from test to test based on the
`
`pathogen to be detected. Thus, manufacturing of test kits and devices for the practice of the
`
`methods could be drastically simplified.
`
`[0004] The methods may be incorporated into any test format or device suitable for the practice of
`
`the methods. Also provided are kits, reagents, etc. for the practice of the methods.
`
`10
`
`15
`
`20
`
`25
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`[0005] Further objectives and advantages of the present invention will become apparent as the
`
`description proceeds. To gain a fill appreciation of the scope of the present invention, it will be
`
`further recognized that various aspects of the present invention can be combined to make desirable
`
`embodiments ofthe invention.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0006] FIGURE 1 depicts a schematic of an exemplary embodiment of the use of ribosomal
`
`amplification to detect Streptococcus A bacteria.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0007] Unless defined otherwise above, all technical and scientific terms used herein have the same
`
`meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
`
`Where a term is provided in the singular, the inventor also contemplates the plural of that term. The
`
`nomenclature used herein and the procedures described below are those well known and commonly
`
`employed in the art.
`
`[0008] The term “amino acid” is intended to embrace all molecules, whether natural or synthetic,
`
`which include both an amino functionality and an acid functionality and capable ofbeing included
`
`in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-
`
`occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having
`
`variant side chains; and all stereoisomers of any of any of the foregoing. The names of the natural
`
`amino acids are abbreviated herein in accordance with the recommendations of lUPAC—IUB.
`
`[0009] The term “antibody” refers to an immunoglobulin, derivatives thereof which maintain
`
`specific binding ability, and proteins having a binding domain which is homologous or largely
`
`homologous to an immunoglobulin binding domain. These proteins may be derived from natural
`
`sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal.
`
`The antibody may be a member of any immunoglobulin class, including any of the human classes:
`
`lgG, IgM, lgA, lgD, and lgE. In exemplary embodiments, antibodies used with the methods and
`
`compositions described herein are derivatives of the lgG class.
`
`[0010] The term “antibody fragment” refers to any derivative of an antibody which is less than filll-
`
`length. In exemplary embodiments, the antibody fragment retains at least a significant portion of
`
`the filll—length antibody’s specific binding ability. Examples of antibody fragments include, but are
`
`not limited to, Fab, Fab’, F(ab’)2, scFv, Fv, dst diabody, and Fd fragments. The antibody
`
`fragment may be produced by any means. For instance, the antibody fragment may be
`
`enzymatically or chemically produced by fragmentation of an intact antibody, it may be
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`recombinantly produced from a gene encoding the partial antibody sequence, or it may be wholly or
`
`partially synthetically produced. The antibody fragment may optionally be a single chain antibody
`
`fragment. Alternatively, the fragment may comprise multiple chains which are linked together, for
`
`instance, by disulfide linkages. The fragment may also optionally be a multimolecular complex. A
`
`filnctional antibody fragment will typically comprise at least about 50 amino acids and more
`
`typically will comprise at least about 200 amino acids.
`
`[0011] The terms “comprise” and “comprising” is used in the inclusive, open sense, meaning that
`
`additional elements may be included.
`
`[0012] The term “including” is used herein to mean “including but not limited to”. “Including”
`
`and “including but not limited to” are used interchangeably.
`
`[0013] The term “mRNA” refers to messenger RNA, or the RNA that serves as a template for
`
`protein synthesis in a cell. The sequence of a strand of mRNA is based on the sequence of a
`
`complementary strand of DNA comprising a sequence coding for the protein to be synthesized.
`
`[0014] “Nucleic acid” refers to polynucleotides such as deoxyribonueleie acid (DNA), and, where
`
`appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents,
`
`analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment
`
`being described, single (sense or antisense) and double-stranded polynucleotides. ESTs,
`
`chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that may be
`
`referred to as nucleic acids.
`
`[0015] The term “pathogen” refers to any organism which may cause disease in a subject, such as a
`
`bacterium, filngus, parasite, virus, etc.
`
`[0016] “Protein” (if single-chain), “polypeptide” and “peptide” are used interchangeably herein
`
`when referring to a gene product, e. g., as may be encoded by a coding sequence. When referring to
`
`“polypeptide” herein, a person of skill in the art will recognize that a protein can be used instead,
`
`unless the context clearly indicates otherwise. A “protein” may also refer to an association of one
`
`or more polypeptides.
`
`[0017] The term “sample” refers to any sample potentially containing pathogens containing
`
`ribosomes.
`
`[0018] The term “ribosomal RNA” or “rRNA” refers to the RNA component of ribosome subunits.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Ribosomes and their subunits are described filrther below.
`
`[0019] Provided in one aspect is a method, comprising:
`
`[0020]
`
`(a)
`
`contacting a support comprising a binding agent capable of binding a
`
`ribosome with a sample comprising ribosomes;
`
`[0021]
`
`(b)
`
`washing the support after contact with the sample;
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`[0022]
`
`(c)
`
`incubating the support in the presence of a solution comprising mRNA and
`
`amino acids under conditions suitable to allow ribosomes bound to the binding agent to translate the
`
`mRNA in the solution into a polypeptide; and
`
`[0023]
`
`(d)
`
`determining the presence of the polypeptide.
`
`5
`
`[0024]
`
`In certain embodiments, the ribosomes are specific to a pathogen and the method fithher
`
`comprises determining the presence of the pathogen in the sample based on the presence of the
`
`polypeptide.
`
`[0025] Provided in another aspect is a method, comprising:
`
`[0026]
`
`(a)
`
`capturing ribosomes on a support using a binding agent capable of binding the
`
`l 0
`
`ribosomes;
`
`[0027]
`
`(b)
`
`incubating the captured ribosomes with a solution comprising mRNA and amino
`
`acids; and
`
`[0028]
`
`(c)
`
`detecting the polypeptide produced by the incubated, captured ribosomes.
`
`[0029] Ribosomes are ribonucleoproteins which are present in both prokaryotes and eukaryotes.
`
`15
`
`They comprise about two-thirds RNA and one-third protein. Ribosomes are the cellular organelles
`
`responsible for protein synthesis. During gene expression, ribosomes translate the genetic
`
`information encoded in a messenger RNA into protein (Garrett et al. (2000) “The Ribosome:
`
`Structure, Function, Antibiotics and Cellular Interactions,” American Society for Microbiology,
`
`Washington, DC)
`
`20
`
`[0030] Ribosomes comprise two nonequivalent ribonucleoprotein subunits. The larger subunit
`
`(also known as the “large ribosomal subunit”) is about twice the size of the smaller subunit (also
`
`known as the “small ribosomal subunit”). The small ribos omal subunit binds messenger RNA
`
`(mRN A) and mediates the interactions between mRN A and transfer RNA (tRNA) anticodons on
`
`which the fidelity of translation depends. The large ribosomal subunit catalyzes peptide bond
`
`25
`
`formation—-the peptidyl-transferase reaction of protein synthesis--and includes (at least) two
`
`different tRNA binding sites: the A—site which accommodates the incoming aminoacyl-tRNA,
`
`which is to contribute its amino acid to the growing peptide chain, and the P—site which
`
`accommodates the peptidyl-tRNA complex, i.e., the tRNA linked to all the amino acids that have so
`
`far been added to the peptide chain. The large ribosomal subunit also includes one or more binding
`
`30
`
`sites for G—protein factors that assist in the initiation, elongation, and termination phases of protein
`
`synthesis. The large and small ribosomal subunits behave independently during the initiation phase
`
`of protein synthesis; however, they assemble into complete ribosomes when elongation is about to
`
`begin.
`
`[0031] Accordingly, as used herein, the term “ribosome” refers to a complex comprising a large
`
`35
`
`ribosomal subunit and small ribosomal subunit. The large ribos omal subunit and small ribosomal
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`subunit are 50 S and 30 S subunits respectively in bacteria and 60 S and 40 S subunits respectively
`
`in eukaryotes.
`
`[0032] Protocols describing the preparation of samples comprising ribosomes are available in the
`
`literature and can be adapted Where needed by those skilled in the art. For example, the preparation
`
`of ribosomes from bacteria can be done essentially as described by Youmans and Youmans, 1965
`
`and adapted as described by Gregory et al., 1983. In general, but particularly when using virulent
`
`Microbes (pathogenic), is recommended to kill the cells prior to further use, for example by
`
`treatment with formalin as described by Michalek and McGhee, 1977, and adjust concentrations to
`
`108 bacterial or fungal cells/ml or 107 protozoa/ml. The preparation can be established to be sterile
`
`when no multiplication occurs upon inoculation on Sheep blood and Mitis Salivarius agars (DIFCO)
`
`or other adapted rich culture medium. Aliquots are stored at -80.degree. C. Subsequently they are
`
`thawed rapidly at 37.degrees. C., and l g of whole cells is re—suspended with l g of micro—glass
`
`beads (0.17—0.18 m) in 1 ml of PMB to which 3 .mu.g/ml Dnase (SIGMA) is added. The cells are
`
`disrupted by shaking for three 2—minute cycles in a Braun homo genizer. lntact cells and debris are
`
`removed by two centrifugations (27.000.times. g followed by 47.000.times. g; 10 minutes each).
`
`[0033] Preparation of ribosomes from fungi and protozoa follow essentially the same procedure but
`
`require adaptation of culture conditions and lysis methods. Given that culture conditions of
`
`cultivatable pathogenic microbes are widely available in published literature, preparation of
`
`ribosomes from such microbes is well within the possibilities of a person skilled in the art.
`
`[0034]
`
`Integrity of the ribosomal subunits is important.
`
`In particular the stabilization of enclosed
`
`large ribosomal RNA’s by divalent cations such as provided by MgClz, concentration which may
`
`need adaptation depending on the microbe and extraction protocol methods used. The ribosomes in
`
`the supernatant can be harvested by centrifugation at 180.000 to 250.000.times. g for 2 to 3 hr and
`
`then subjected to 5 successive washes in PMB at 180.000 to 250.000.times. g for 2 to 3 hr each. The
`
`ribosomal preparation is then clarified twice by two 20-min. centrifugations at 47.000.times. g and
`
`the supernatant is filtered through a sterile 0.45 micron Millipore filter (Millipore Filter Corp.).
`
`Non—dissociated (=intact) ribosomes can be prepared from gram-negative, Rnase-minus mutant
`
`bacteria such as Escherichia coli MRE600 following the method of Staehilin et. al., 1969, with
`
`modifications as described by M. M. Yusupov and A. S. Spirin. 1988. The preparations can then
`
`adjusted to, for example, 20 mg/ml on the basis of protein content by standard protein quantification
`
`methods, using, for example, bovine serum albumin as a standard, and maintained at -80.degree. C.
`
`until used. Characterization of the rib osomal fraction and purity can be determined by spectral
`
`analysis at 235, 280 and 260 nm in order to determine the contamination of rib osomal RNA by
`
`DNA polyacrylamide gel electrophoresis permits to evaluate the presence of ribosomal proteins and
`
`potential contaminating proteins. The degree of intactness can be evaluated by loading a sample of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`the original homogenate onto a 10% to 40% sucrose gradient, containing an appropriate
`
`concentration of Mg C12 and centrifugation. The elusion profile of the sucrose gradient Will show
`
`the different fractions: 100$=dimers of 708 ribosomes, 7OS=intact ribosomes, 6OS=interacting SOS
`
`and 308 ribosomal subunits, SOS=large ribosomal subunit, 3OS:small ribosomal subunit, material
`
`less than 3OS=degradation products and contaminants. In good preparations that target non—
`
`dissociated ribosomes, the 708 peak contains over 80% of all material. Optionally, the 708 peak
`
`containing the target non-dissociated ribosomes may constitute at least 50%, 60%, 70% or 90% of
`
`all material.
`
`[0035] Pathogens that may be detected using the above methods include any organism comprising
`
`ribosomes. Organisms from Which ribosomes may be isolated include, but are not limited to, the
`
`following:
`
`[0036] Ribosomes from bacteria such as: Acinetobacter calcoaceticus, A. haemolyticus,
`
`Aeromonas hydrophilia, Bacteroidesfragilis, B. dislasonis, Bacteroides 3452A homology group, B.
`
`vulgatus, B. ovalus, B. thetaiotaomicron, B. uniformis, B. eggerthii, B. splanchnicus, Branhamella
`
`catarrhalis, Campylobacterfetus, C. jejuni, C. coli, Citrobacterfreundii, Clostridium difficile, C.
`
`diphtheriae, C. ulcercms, C. accoleris, C. afermentans, C. amycolatum, C. argentorense, C. auris. C.
`
`bovis, C. confusum, C. coyleae, C. durum, C. falsenii, C. glucuronolyticum, C. imitans, C. jeikeium,
`
`C. kutscheri, C. kroppenstedtii, C. lipophilum, C. macginleyi, C. matruchoti, C. mucifaciens, C.
`
`pilosum, C. propinquum, C. renale, C. riegelii, C. sanguinis, C. singulare, C. striatum, C.
`
`sundsvallense, C. thomssenii, C. urealyticum, C. xerosis, Enterobacter cloacae, E. aerogenes,
`
`Enterococcus avium, E. casseliflavus, E. cecorum, E. dispar, E. durans, E. faecalis, E. faecium, E.
`
`flavescens, E. gallinarum, E. hirae, E. malodoralus, E. mundlii, E. pseudoavium, E. raffinosus, E.
`
`solitarius, Francisella tularensis, Gardnerella vaginalis, Helicobacter pylori, Kingella dentrificans,
`
`K. kingae, K. oralis, Klebsiella pneumoniae, K oxytoca, Moraxella catarrhalis, M atlcmtae, M
`
`lacunata, M nonliquefaciens, M osloensis, M phenylpyruvica, Morganella morgcmii,
`
`Parachlamydia acanthamoebae, Pasteurella multocida, P. haemolytica, Proteus mirabilis, Proteus
`
`vulgaris, Providencia alcalifaciens, P. rettgeri, P. stuartii, Serratia marcescens, Simkania
`
`negevensis, Streptococcus pneumoniae, S. agalactiae, S. pyogenes, Treponema pallidum, Vibrio
`
`cholerae, and V. parahaemolyticus.
`
`[0037] Ribosomes from facultative intracellular bacteria such as: Bordetella pertussis, B.
`
`parapertussis, B. bronchiseptica, Burkholderia cepacia, Escherichia coli, Haemophilus
`
`actinomycetemcomitans. H. aegyptius. H. aphrophi/us. H. ducreyi. H. felis, H. haemoglobinophilus.
`
`H. haemolyticus, H. influenzae, H. paragallinarum, H. parahaemolyticus, H. parainfluenzae, H.
`
`paraphrohaemolyticus, H paraphrophilus, H. parasuis, H. piscium, H. segnis, H somnus, H.
`
`vaginalis, Legionella adelaidensis, L. anisa, L. beliardensis, L. birminghamensis, L. bozemcmii, L.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`brunensis, L. cherrii, L. cincinnatiensis, Legionella drozanskii L. dumoflli, L. erythra, L.
`
`fairfieldensis, L. fallonii, L. feeleii, L. geestiana, L. gormanii, L. gratiana, L. gresilensis, L.
`
`hackeliae, L. israelensis, L.j0rdanis, L. lansingensis, Legionella landiniensis L. longbeachae,
`
`Legionella lytica L. maceacherhii, L. micdadei, L. moravica, L. hautarum, L. oakridgehsis, L.
`
`5
`
`parisiensis, L. pittsburghensis, L. pneumophila, L. auateirensis, L. quinlivanii, L. rowbothamii, L.
`
`rubrilucens, L. sainthelensi, L. santicrucis, L. shakespearei, L. spiritensis, L. steigerwaltii, L.
`
`taurinensis, L. tucsonensis, L. wadsworthii, L. waltersii, L. worsleiensis, Listeria denitrificans, L.
`
`grayi, L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri, L. welshimeri, Mycobacterium
`
`abscessus, M. africanum, M. agri, M. aichiense, M alvei, M asiaticum, M aurum, M.
`
`10
`
`austroafricanum, M. avium, M bohemicum, M. bovis, M branderi, M. brumae, M celatum, M.
`
`chelonae, M chitae, M chlorophenolicum, M chubuense, M confluentis, M conspicuum, M
`
`coo/(ii, M diernhoferi, M doricum, M duvalii, M elephantis, M fallax, M farcinogenes, M
`
`flavescens, M forluilum, M frederiksbergense, M gadium, M gastri, M genavense, M gilvum, M
`
`goodii, M. gordonae, M. haemophilum, M. hassiacum, M. heckeshornense, M. heidelbergense, M.
`
`15
`
`hiberniae, M immunogenum, M intracellulare, M interjectum, M intermedium, M kansasii, M
`
`komossense. M kubicae. M lentiflavum. M leprae. M lepraemurium, M luteum, M
`
`madagascariense, M mageritense, M malmoense, M marinum, M microti, M moriokaense, M
`
`mucogenicum, M murale, M neoaurum, M nonchromogenicum, M novocastrense, M obuense, M
`
`parqfortuitum, M paratuberculosis, M peregrinum, M phage, M phlei, M porcinum, M
`
`20
`
`poriferae, M pulveris, M rhodesiae, M scrofulaceum, M serzegalense, M septicum, M shimoidei,
`
`M simiae, M smegmatis, M sphagni, M szulgai, M terrae, M thermoresistibile, M tokaiense, M
`
`triplex, M lriviale, M tuberculosis, M lusciae, M ulcerans, M vaccae, M wolinskyi, M xenopi,
`
`Neisseria animalis, N. canis, N. cinerea, N. denitrificans, N. dentiae, N. elongata, N. flava, N.
`
`flavescens, N. gonorrhoeae, N. iguanae, N. lactamica, N. macacae, N. meningitidis, N. mucosa, N.
`
`25
`
`ovis. N. perflava. N. pharyngis var. flava. N. polysaccharea. N. sicca. N. subflava. N. weaveri,
`
`Pseudomonas aeruginosa, P. alcaligenes, P. chlororaphis, P. fluorescens, P. luteola, P. mendocina,
`
`P. monteilii, P. oryzihabitans, P. pertocinogena, P. pseudalcaligenes, P. putida, P. stutzeri,
`
`Salmonella bacteriophage, S. bongori, S. choleraesuis, S. enterica, S enteritidis, S. paratyphi, S.
`
`typhi, S. typhimurium, S. typhimurium, S. typhimurium, S. typhimurium bacteriophage, Shigella
`
`30
`
`boydii, S. dysenteriae, S. flexneri, S. sonnei, Staphylococcus arlettae, S. aureus, S. auricularis, S.
`
`bacteriophage, S. capitis, S. caprae, S. carnosus, S. caseolyticus, S. chromogenes, S. cohnii, S.
`
`a’elphini, S. epidermidis, S. eauorum, S. felis, S. fleurettii, S. gallinarum, S. haemolyticus, S.
`
`hominis, S hyicus, S intermedius, S. kloosii, S lentus, S. lugdunensis, S lutrae, S muscae, S
`
`mutans, S. pasteuri, S. phage, S. piscifermentans, S pulvereri, S. saccharolyticus, S saprophyticus,
`
`35
`
`S. schleiferi, S. sciuri, S. simulans, S succinus, S. vitulinus, S. warneri, S. xylosus, Ureaplasma
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`urealyticum, Yersinia aldovae, Y. bercovieri, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y.
`
`kristensenii, Y mollaretii, Y. pestis, Y. philomiragia, Y. pseudotuberculosis, Y. rohdei, and Y.
`
`ruckeri.
`
`[0038] Ribosomes from obligate intracellular bacteria, such as: Anaplasma bowls; A. caudatum, A.
`
`centrale, A. marginale A. ovis, A. phagocytophila, A. plalys, Bartonella bacilliform is, B.
`
`clarridgeiae, B. elizabethae, B. henselae, B. henselae phage, B. quintana, B. taylorii, B. vinsonii,
`
`Borrelia afzelii, B. andersonii, B. anserina, B. bissettii, B. burgdmferi, B. crocidurae, B. garinii, B.
`
`hermsii, B. japom'ca, B. miyamotoi, B. parkerz', B. recurrentis, B. Iurdz', B. turicatae, B. valaz'sz'ana,
`
`Brucella abortus, B. melitensis, Chlamydia pneumoniae, C. psittaci, C. trachomatis, Cowdria
`
`ruminantium, Coxiella burnetii, Ehrlichia cams, E. chaffeensis, E. equi, E. ewingii, E. mum's, E.
`
`phagocytophila, E. platys, E. risticii, E. ruminantium, E. sennetsu, Haemobartonella canis, H. felis,
`
`H. muris, Mycoplasma arthriditis, M. buccale, M. faucium, M fermentans, M. genitalium, M
`
`hominis, M laidlawii, M [ipophilum M orale, M penetrans, M pirum, M pneumoniae, M
`
`salivarium, M. spermatophilum, Rickettsia australis, R. conorii, R. felis, R. helvetica, R. japonica,
`
`R. massilz'ae, R. montanensz's, R. peacockiz', R. prowazekii, R. rhipz’cephali, R. rickettsz'i, R. Sibirz'ca,
`
`and R. lyphi.
`
`[0039] Ribosomes from facultative intracellular fungi, such as: Candida Candida aaseri, C.
`
`acidothermophilum, C. acutus, C. albicans, C. anatomiae, C. apis, C. apis var. galacta, C.
`
`atlantica, C. atmospherica, C. auringiensis, C. bertae, C. berrhtae var. chiloensis, C. berrhetii, C.
`
`blankii, C. boidinii, C. boleticola, C. bombi, C. bombicola, C. buinensis, C. bulyri, C. cacaoi, C.
`
`cantarellii, C. cariosilignicola, C. castellii, C. castrensis, C. catenulata, C. chilensis, C.
`
`chiroplerorum, C. coipomensis, C. dendronema, C. deserlicola, C. diddensiae, C. diversa, C.
`
`entomaea, C. entomophila, C. ergatensis, C. ernobii, C. ethanolica, C. ethanothermophilum, C.
`
`famata, C. fluviotilis, C. fragariorum, C. fragicola, C. friedrz'chii, C. fructus, C. geochares, C.
`
`glabrata, C. glaebosa, C. gropengiesseri. C. guilliermondii, C. guilliermondii var. galactosa, C.
`
`guilliermondii var. soya, C. haemulom'i, C. halophila/C. versatilis, C. holmii, C. humilis, C.
`
`hydrocarbofumarica, C. inconspicua, C. insectalens, C. insectamans, C. intermedia, C. javanica, C.
`
`kefyr, C. krissii, C. krusei, C. krusoides, C. lambica, C. [usiraniaa C. magnoliae, C. maltosa, C.
`
`mamillae, C. mariS, C. maritima, C. melibiosica, C. melinii, C. methylica, C. milleri, C. mogii, C.
`
`molischiana, C. montana, C. multiS-gemmis, C. musae, C. naeodendra, C. nemodendra, C.
`
`nitratophila, C. norvegensis, C. nowegica, C. oleophila, C. oregonensis, C. osornensis, C.
`
`paladigena, C. parapsi/osis, C. pararugosa, C. periphe/osum, C. petrolmensis, C. petrophflum, C.
`
`philyla, C. pignaliae, C. pinrolopesii var. pintolopesii, C. pintolopesii var. Sloofliae, C. pinus, C.
`
`polymorpha, C. populi, C. pseudointermedia, C quercitrasa, C. railenensis, C. rhagii, C.
`
`rugopelliculosa, C. rugosa, C. sake, C. salmanticensis, C. savom'ca, C. sequanensis, C. Shel/tame, C.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`Silvae, C. Silvicultrix, C. solam', C. sonorensis, C. sorbophila, C. Spandovensis, C. Sphaerica, C.
`
`stellata, C. succiphila, C. termis, C. terebra, C. tropicalis, C. utilis, C. valida, C. vanderwaltii, C.
`
`vartiovaarai, C. veronae, C. vini, C. wickerhamii, C. xestobii, C. zeylanoides, and Hisroplasma
`
`capsulatum.
`
`[0040] Ribosomes from obligate intracellular protozoans, such as: Brachiola vesicularum, B.
`
`connori, Encephalitozoon cuniculi, E. hellem, E. intestinalis, Enterocytozoon bieneusi, Leishmania
`
`aethiopica, L. amazonensis, L. braziliensis, L. chagasi, L. d0720vani, L. donovani chagasi, L.
`
`donovani donovam', L. donovani infantum, L. enriettiz', L. guyanensis, L. infantum, L. major, L.
`
`mexiccma, L. panamensis, L. peruviana, L. pifanoi, L. tarentolae, L. tropica, Microsporidium
`
`ceylonensis, M. africanum, Nosema connori, N. ocularum, N. algerae, Plasmodium berghei, P.
`
`brasilianum, P. chabaudi, P. chabaudi adami, P. chabaudi chabaudi, P. cynomolgi, P. falciparum,
`
`P. fragile, P. gallinaceum, P. knowlesi, P. [ophuraa P. malariae, P. ovale, P. reichenowi, P.
`
`Simiovale, P. simium, P. vinckeipelleri, P. vinckei vinckei, P. vivax, P. yoelii, P. yoelii nigeriensis,
`
`P. yoelii yoelii, Pleistophom anguillarum, P. hippoglossoideos, P. mirandellae, P. ovariae, P.
`
`lypz'calis, Septata intestinalis, Toxoplasma gondz'i, Trachz'plez'stophom hominis, T. anthropophthem,
`
`Vittafbrma corneae, Trypanosoma avium, T. brucei, T. brucei brucei, T. brucei gambiense. T.
`
`brucei rhodesiense, T. cobitis, T. congolense, T. cruzi, T. cyclops, T. equiperdum, T. evansi, T.
`
`dionisii, T. godfreyi, T. grayi, T. lewisi, T. mega, T. microti, T. pestanai, T. rangeli, T. rotatorium, T.
`
`Simiae, T. theileri, T. varani, T. vespertilionis, and T. Vivax.
`
`[004]] The ribosomes from the pathogen may be bound to the support, i.e., immobilized, Via a
`
`binding agent. Immobilizing complexes such as ribosomes to a support is well Within the skill of
`
`one in the art. In certain embodiments, the binding agent is capable of specifically binding a
`
`ribosome, e. g. is specific for a ribosome from a particular pathogen. For example, the binding agent
`
`may be a polyclonal or monoclonal antibody or an antibody fragment, e. g. specific for the ribosome
`
`to be captured. For example, the antibody or antibody fragment may be specific for a ribosomal
`
`protein comprising the ribosome. In other embodiments, the binding agent is a ribosomal binding
`
`protein specific for the ribosome to be captured. In other embodiments, the binding agent is capable
`
`of binding ribosomal RNA, and may be a nucleic acid, i. e., a nucleic acid complementary to and
`
`specific for the ribosomal RNA of the ribos ome to be captured.
`
`In still other embodiments, the
`
`binding agent is a protein that binds ribos omal RNA, e. g., an RNA binding protein.
`
`[0042] The support may be any suitable material for immobilizing ribosomes via any of the above-
`
`described binding agents. In certain embodiments, the support is a porous material, e. g.
`
`nitrocellulose. In other embodiments, the support is a particle, e. g., a magnetic particle. In certain
`
`embodiments, the support is comprised of a plurality of particles.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`[0043]
`
`In certain embodiments, the support is part of a test strip or test chamber. In some
`
`embodiments, the support is a test strip. In other embodiments, the support is part of a lateral flow
`
`device. In still other embodiments, the support is resin or other material suitable for packing in a
`
`column.
`
`In still other embodiments, the support is a plate with wells or a test tube.
`
`[0044] The support may be washed with any buffer appropriate to maintaining the biological
`
`function of the ribosomes yet sufficient to remove unbound ribosomes and other material. The
`
`wash buffer must contain suitable inhibitors of ribosomal activity in order to ensure the residual
`
`ribosomes do not translate mRNA. Alternatively, or in addition to adding inhibitors, the wash
`
`buffer may be autoclaved to destroy ribosomal activity. Appropriate pH, glycerol or other
`
`stabilizing molecules such as ligands, polyethylene glycol, etc., and the presence or absence of
`
`reducing agent, chelating agent, cofactors, detergents, protease inhibitors, ribosomal activity
`
`inhibitors are accordingly all important considerations. The support may be washed with anywhere
`
`from about 5 to 20 volumes of each wash buffer to eliminate unbound ribosomes from the support,
`
`e.g., in embodiments wherein the support is or is comprised within a column, lateral flow, or other
`
`format wherein the wash may flow over the support. In certain embodiments wherein the support is
`
`a particle, the washing may comprise moving the particles from one liquid, e. g., the sample, to
`
`another liquid, e. g., the wash.In certain embodiments, the methods described above may be
`
`modified to detect viruses. In the case of RNA viruses, a sample potentially comprising an RNA
`
`Virus may be flowed over a surface comprising antibodies against the virus and ribosomes. As the
`
`sample is flowed over the surface, virus es, if present may be captured by the antibodies. After a
`
`wash step as described above to remove unbound materials, the viruses may be lysed by flowing a
`
`lysis solution over the surface comprising the bound viruses. The lysis releases the RNA, which is
`
`available for translation by the ribosomes as follows.
`
`[0045] After washing, the support is incubated in the presence of a solution comprising mRNA and
`
`amino acids under conditions suitable to allow ribosomes bound to the binding agent to translate the
`
`mRNA in the solution into a polypeptide. In certain embodiments, constant temperature is
`
`maintained during the incubation.
`
`[0046] The solution may include one or more energy sources providing chemical energy for protein
`
`synthesis. Further, the solution includes at least one nucleic acid template, for example, an mRNA.
`
`The solution may include enzymes, translation factors or co—factors. In certain embodiments, the
`
`solution may include E. coli rare t—RNAs selected from tRNAs for amino acids arginine, proline,
`
`glycine, leucine or isoleucine. Further, the solution may include lipids, cholesterol, or membranes.
`
`[0047] The solution may also include an inhibitor of an enzyme that degrades the template or other
`
`enzymes necessary for the translation reaction, such as phosphatases, proteases, nucleas es,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`-10-
`
`

`

`WO 2008/133705
`
`PCT/US2007/082096
`
`deoxyribonucleases, or ribonucleases. Further, it may include an enzyme to catalyze hydrolysis or
`
`formation of phosphodiester bonds.
`
`[0048] Still further, the solution may include at least one molecular chaperone or a foldase. The
`
`molecular chaperone or foldase includes, but is not limited to, GroEL/ES, GroEL, GroES, TF,
`
`DnaK, DnaJ, GrpE, ClpB, kaA, Skp, DsbA, Dst, peptidyl prolyl cis/trans isomerase (PPI),
`
`chaperonin 60, chaperonin 10, TCPl, TF55, heat shock protein 60, Cpn60, heat shock protein 10,
`
`Cpnl 0, Lim protein, or signal recognition particle.
`
`[0049] The amount of protein produced in a translation reaction can be measured by various
`
`means, such as specific enzymatic activity, UV or visible light absorption or fluorescence.
`
`Products of protein synthesis may also be detected by using antibody based assays. Another method
`
`to quantitate the amount of protein produced in a coupled in vitro transcription translation reactions
`
`is to perform the reaction using a known quantity of radiolabeled amino acid such as 35S—methionine
`
`or 3H-leucine and subsequently measuring the amount of radiolabeled amino acid incorporated into
`
`the newly synthesized protein.
`
`lncorporation assays will measure the amount of radiolabeled amino
`
`acids in all proteins produced in an in vitro translation reaction including truncated protein products.
`
`[0050] Other methods for detecting nascent proteins are described in US. Published Patent
`
`Application 20050032078.
`
`[0051] For use in the diagnostic and other applications suggested above, kits and devices for the
`
`practice of the above—described methods are also provided. Devices for practice of the methods
`
`include lateral flow devices (wherein the reagents employed in the reaction may be dried onto the
`
`chromatographic support