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Mat Kadi Tora Tutti Tutto Ultima Hora En Lithuania MAT KADI TORA TUTTI USTUTTO 20180148498A1 ULTIMAHORA EN LITHUANIA ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 /0148498 A1 Kozel et al. (43 ) Pub . Date : May 31 , 2018 ( 54 ) FUNGAL DETECTION USING MANNAN Publication Classification EPITOPE (51 ) Int. Cl. @(71 ) Applicant: BOARD OF REGENTS OF THE COZK 16 / 14 (2006 .01 ) NEVADA SYSTEM OF HIGHER GOIN 33 /569 ( 2006 . 01) EDUCTION , ON BEHALF OF THE CO7K 16 / 44 ( 2006 . 01 ) UNIVERSITY OF NEVADA , RENO , (52 ) U . S . CI. NV (US ) CPC . .. .. CO7K 16 / 14 ( 2013 .01 ) ; GOIN 33 /56961 ( 2013 .01 ) ; COZK 2317/ 622 (2013 . 01 ) ; COOK @(72 ) Inventors: Thomas R . Kozel , Reno , NV (US ) ; 2317 /33 (2013 . 01 ) ; CO7K 2317/ 92 ( 2013 .01 ) ; Breeana HUBBARD , Pullman , WA (US ) ; Amanda CO7K 16 /44 ( 2013 .01 ) BURNHAM -MARUSICH , Reno , NV (US ) ( 57 ) ABSTRACT ( 21) Appl . No. : 15 /567 , 547 (22 ) PCT Filed : Apr. 23 , 2016 Non - invasive methods are provided herein for diagnosing samples as including a fungus , including fungal infection or ( 86 ) PCT No. : PCT/ US16 /29085 contamination , with specific monoclonal antibodies capable $ 371 ( c) ( 1 ), of detecting molecules associated with fungi in the sample , ( 2 ) Date : Oct. 18 , 2017 such as a biological or environmental sample . These mol ecules can be identified using various methods, including Related U . S . Application Data but not limited to antibody based methods , such as an ( 60 ) Provisional application No. 62 /151 , 865, filed on Apr . enzyme- linked immunosorbant assay (ELISA ) , or a lateral 23 , 2015 . flow immunoassay . Limit of detection (nam ) Vucor Aspergillus 540 Ugariun Reciprocaltiter . Candida 16:55555555555555555555555555555555555555 öö Mucor Aspergillus Fusarum Candida Patent Application Publication May 31 , 2018 Sheet10f8 US 2018/ 0148498Al ????????????????????????????????????????????????????????????????????????????? ??_ ??? f ' 9 ? ? ? ????? ?????$ ? / 28; & ? ? {QK ? Reciprocaltiter é 4 ????????????????????????????????????????????????????????????????????????????????????????????????????????2 é _é AHATARCH Aspergillus Fusarium Candida FIG1 Patent Application Publication May 31 , 2018 Sheet 2 of 8 US 2018 / 0148498 A1 mAb 2DA6 DNA Sequence Light chain : DNA sequence ( 384 bp ) (SEQ ID NO : 1 ) Leader sequence ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATTATGTCCAGAGGA C FR1 AAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGCAGAAAGTCACCATAACCTG CDR1 FR2 ? AGTGTCAGCTCAAACATACATTTCATGCAC TGGTACCAGCAGAAGTTAGGATTc?ccccCAAACTC CDR2 TGGATTTAT GACACATCCAAACTGACTCCT GGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGA FR3 CCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGC CATCAGTG CDR3 FR4 GAGTAGTCACCCACATACG TTCGGATCGGGGACCAAGCTGGAAATAAAA Heavy chain : DNA sequence ( 408 bp ) (SEQ ID NO : 2 ) Leader sequence ATGGATTGGACCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCC CAGGTTCAGC FR1 TGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAGGCTTTCCTGCAAGACTACTGG CDR1 FR2 CTACACATTCACT GGCAACTGGATTGAG TGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATT CDR2 GGA GAGATCTTACCTGGAAATAGTCGGACTAATTATAATGAGAAGTTTAAGGGC AAGGCCACTTTCA FR3 CTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACACCTGAAGACTCTGCCAT CDR3 FR4 CTATTTCTGTGCAAGA GTGACTGGGACGTCCTTTGACTACT GGGGCCAAGGCACCACTCTCACAGTC TCCTCT FIG . 2A Patent Application Publication May 31 , 2018 Sheet 3 of 8 US 2018 / 0148498 A1 mAb 2DA6 Amino Acid Sequence Light chain : Amino acids sequence ( 128 AA ) (SEQ ID NO : 3 ) 1 Leader sequence FR1 1 CDR1 FR2 MDFQVQIFSFLLISASVIMSRG QIVLTQSPAIMSASPGQKVTITC SVSSNIHFMH WYQQKLGFSPK LCDR21 FR3 1 CDR3 I FR4 1 LWIY DTSKLTP GVPARFSGSGSGTSYSLTISSMEAEDAASYFC HQWSSHPHT FGSGTKLEIK Heavy chain : Amino acids sequence ( 136 AA ) (SEQ ID NO : 4 ) 1 Leader sequence | FR1 | CDR11 FR2 MDWTWVFLFLLSVTAGVHS QVQLQQSGAELMKPGASVRLSCKTTGYTFT GNWIE WIKQRPGHGLEWI CDR2 FR3 | CDR3 1 FR4 G EILPGNSRTNYNEKFKG KATFTADTSSNTAYMOLSSLTPEDSAIYFCAR VTGTSFDY WGQGTTLT VSS FIG . 2B Patent Application Publication May 31 , 2018 Sheet 4 of 8 US 2018 / 0148498 A1 Clone 11C16 Light chain (SEQ ID NO : 5 ) < < - - - - - - -- -— —- - - - —- —- - - - -- -- - - - —- —- - - - - - - - - - - FR1 - IMGT -- - - - - -- - - —- -— —- - -- - - gagct g t g gactcaqtcgccctct . acatct Ccgcccta ggagcctcggccaag von monen mensen montere mom mom moet moet saam met meer nou wo mom mom moet ons >> CDR1 - IMGT < - - - - - - - .. .. .. 10tca 1 cda . accta cacca t t www .. .. FR2 _ IMGT - - - - - wwwwwwwwwww - CDR t a Bacaace gagga t ata 03 c aacttaa ga . 2 - IMGT - - - - - - - - - - - - - - - - - - - - - - - - |- - - - - - - - - - - - - - - - - - - - . .. .. ggaacctaggaacctacaccaaagagaccggtgtccct caccaa g a cct . gatcgcttctcgggctccago 1 - - - FR3 - IMGT - - - - - I I - . .. totctggggctgaccgctacttgatcatctccagcgtccaggttgatgacgaggcc tact tcatctcc rt a - - - -- - |- - - - - - - > CDR3 - IMGT o IOctacta at rt ttattc ggcggagagaccc 0) c rt g Heavy chain (SEQ ID NO : 6 ) V< - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FR1 - IMGT - - - - - - - - - - . - - CDR1 - IMGT < - - - - - . tca 0 caaagcct tagattctccctcgal LCCCCCCC . aqtgactactaaataaac - - - - - - - - - - - - - - - FR2 - IMGT - - - - - - - - - - - - - - - - - - - > CDR 0 g tccagaga ID gaaccattaqta ctaat . .2 -. IMGT. ggtagcacatactacacgagctgggcgaaa- - - - - - - - - - - - - - - -- - - - - .- -. ggccgattcaccatctccaaa- - - 1 - - -- - FR3 _ IMGT - acct g a CC . ac 10 C a a g a a 10 c gagga a C - - - - - T- - - - > CDR3 - IMGT D acctattID anel 0st Cca agaacatatatt gat atttct gggc FIG . 3A Patent Application Publication May 31 , 2018 Sheet 5 of 8 US 2018 / 0148498 A1 Clone 9M12 Light Chain (SEQ ID NO : 7 ) V< .- .. .. -. -. -. -. - - - - - - - - - - - - - - - - - - - FR1 - IMGT - - - - - - .. gaga . cagctggtggagtccgggggt . cgcctggtcacgcctgggacacccctgacaa g a * * * * na nova con una nuova au -- > CDR1 - IMGT - - -- - - - - - ctcgcctgcacagcctctggattctccctct ca acct CCTC . .. .. .. agtagccacgacatgatctc I | | - - - - - - FR2 - IMGT I | - L CDR tgggtccgccaggctccaggggagggactggaatacatcggatacattactgctggtg X rt catta . < - - - - - - - - T - - - - - - - - - - - - - - - - - - - - - - - - - - v at tutututututute - 2 - IMGT Hitta tutta . ggtagcccatactacgcgagctgggcaaaa- - . ggccgattcaccatctccaga- wwwwwwww 23 - IMGT 1 ** * * * * www ww www www www * * * * * * www wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww accacctcgacc a . acggtggatctgaaaatggccagtctgacaaccgagacacggccaa c t ac - - - - - - - - > cgtatttctg Sequence cut short - no CDR3 identified Heavy chain (SEQ ID NO : 8 ) < - - - - - - - - - - - -- - -- - - - - - - - - - - - - - - - FR1 - IMGT - - - . ccctgaca -- - -* -* --* -* we wou wou vou mouww - - o- ou o noso sou wou now. - - -- > CDR1 - IMGT < - - - -ou nou mode ctcgcctgcacagcctctggattctccctct a CCCCC . .. .. agtagocacgacatgatocca t 1 I | - | | CDR tgggtccgccaggctccaggggagggactggaatacatcggatacattactgctggtUT g ta > atta . 2 - IMGT - |- 1- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . ggtagcccatactacgcgagctgggcaaaaCccatac g c aaa . .. ggccgattcaccatctccagatca CCat ct 1 -- - - - - - - - FR3 - IMGT - - - - - - - -- - * * * * www www it with a www * * w ith i with Mr + w w w w www www t 0 c tcgacca CC . acggtggatctgaaaatggccagtctgacaaccgaggacacggccg g a a ??? a d a c - - - - - - - - - - - > CDR3 - IMGT acgtatttctgtggcagaggtgcttattctggttttggttttgacatctggggcccaggc FIG . 3B Patent Application Publication May 31 , 2018 Sheet 6 of 8 US 2018 / 0148498 A1 LODSUMMANRANNANAMAMAR (nomo Wild type 38 Mon 0 . 32 Reciprocaltiter ãogo Reciprocaltiter wwwwwwwwwwwwwwwwwwwwwwwwwww wa! WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW ???the Mnn2 FIG . 4 Reciprocaltiter oano ?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? *hint theother WT-periodate ??y??????** WT-mock M???periodate -I???K Mnn2SET ???late -periodate WT -Proteinase k* WT-mock Mnn2Proteinasek -mock ProteinaseWITH k FIG . 5 Patent Application Publication May 31 , 2018 Sheet 7 of 8 US 2018 / 0148498 A1 FIG . 6 ** * zoKuomiz Basidiorrycota ????????????????????? ?????????????????????????????. ??? wwindidriuwwwwwwwwrive viriki * * * * * NorversitA nnicolai es in wwwwwwwww som ö Reciprocaltiter ö mmmmmmmmmmmmmmmmmmmmmmmmmmmmmm ö nnnnnnnnnwwwwwwwwwwwwwwwwwwwwww Sunwinnnn Rhizopusoryzae Mucom als Deu ameffei Batrachachytrumderdrobat dis sn0000000 jou Malassezia steuSchizosaccharomycesPseudogmascusPneumocystis furfur carina MicrosporumpomkedestructansEpidemiophyton Timagophytes Aspergilluscanis floccosumPencilium fumigatus BotrytisStromatinia Scedosporium cinerea Fusariumcepiola Maghaporthaaposem solaniSaccharomyces Cardida Onze albicans cerevisiae Patent Application Publication May 31 , 2018 Sheet 8 of 8 US 2018 / 0148498 A1 human and veterinary dematophytes Fungi producing combat- induced Wavefga } { { ??17 { ? Negativecontrol07007 $£N # Negativecontrol MaudouintiM.cans 1 Semudabauau Toubra 7?. tonsurans Iviola Rhizopus001111 „ Shradsy Control ???????????????? FIG . 7 Blue stain fungus in pinus Allium white rot Grosmannia clavigera Stromatinta cepivora tittar ETT: : tart 1 e work CE a SA 1 th1528500 tra seasedE Control Test FIG . 8 US 2018 /0148498 A1 May 31 , 2018 FUNGAL DETECTION USING MANNAN fungi , such as from fungal infection or contamination , in a EPITOPE sample or subject . The present invention satisfies this need . CROSS -REFERENCE TO RELATED SUMMARY OF THE INVENTION APPLICATIONS [0007 ] As described herein , the present invention relates 10001 ] The present application claims priority under 35 to compositions and methods for detecting a fungus in a U . S . C . $ 119 ( e ) to U . S .
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