Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids.

RESEARCH ARTICLE Open Access De elopmen o an enzyme linked immunoso ben assay o de ec ion o cya hane di e penoids Tian Shen 1 , Lena M Ho 1 , Heike Hausmann 2 , Ma c S adle 3 and Holge Zo n 1* Abs ac Backg ound: So-called cya hane ype di e penoids a e p oduced as seconda y me aboli es by basidiomyce es. Based on hei an ibac e ial, ungicidal, and cy o oxic p ope ies, cya hane ype e penoids ep esen in e es ing a ge compounds in ungal bio echnology. Resul s: An indi ec compe i i e enzyme linked immunoso ben assay has been de eloped o de ec ion o cya hane ype di e penoids. Rabbi polyclonal an ibodies we e aised agains a mix u e o s ia al A and B conjuga ed o bo ine se um albumin. The condi ions o di ec a achmen o he hap en s ia al B o a solid phase by passi e adso p ion we e op imized. The c oss eac i i ies o he s ia als A, C and D, o he s ia ins A and B, and o he e inacines C and P o s ia al B we e de e mined. The alida ion s udy showed ha he ELISA was p ecise and sensi i e. The a e age IC 50 o s ia al B was 36.0 ng mL −1 wi h an in e -assay coe icien o a ia ion (CV) o 13.2% (n = 5). Reco e ies om s ia al B spiked samples in he assay we e in he ange o 97.3 –125.9%. A good co ela ion be ween he s ia al B concen a ion measu ed by he ELISA and by HPLC-DAD (y = 1.1122× –0.1585, R 2 = 0.9942) was ob ained om linea eg ession analysis. The sui abili y o he ELISA o de ec ion o cya hane ype di e penoids in subme ged cul u es and ui ing bodies o H. e inaceus was s udied. I showed c oss eac i i y wi h supe na an s om subme ged cul u es and ex ac s he eo , bu did no show c oss eac i i y wi h ex ac s om ui ing bodies. Conclusions: The de eloped me hod is app op ia e o quali a i e and quan i a i e de ec ion o cya hane di e penoids in complex mix u es. Due o i s high sensi i i y and speci ici y, i ep esen s an ideal sc eening me hod o disco e ing new cya hane di e penoids and new po en ial p oduce s o hem. Keywo ds: Cya hane di e penoids, ELISA, He icium e inaceus, S ia al, E inacine Backg ound Cya hine and allocya hine we e he i s epo ed cya hane di e penoids disco e ed by Allbu and Aye om a s a ic liquid cul u e o he ungus Cya hus helenae (H.J.B odie) in he ea ly 1970s [1,2]. They we e p o en o be ac i e agains ac inomyce es, G am-posi i e and G am-nega i e bac e ia, and some ungi, including de ma ophy es. A e - wa ds, a ious s uc u ally ela ed compounds, so-called cya hane ype di e penoids, we e isola ed om di e en basidiomyce ous cul u es, e.g. s ia als and s ia ins om Cya hus spp., sa codonins om Sa codon spp., and e ina- cines om He icium spp. (Figu e 1) [3-7]. All o hese compounds sha e a cya hane skele on consis ing o i e-, six-, and se en membe ed ings and possess an ibac e ial, ungicidal, and cy o oxic p ope ies. Fungal ex ac s p e- pa ed om C. s ia us showed signi ican inhibi o y e ec s on he NF-κB ac i a ion pa hway and migh be applied o cance he apeu ics [8]. E inacines p omo e ne e g ow h ac o (NGF) syn hesis, which sugges s he applica ion o H. e inaceus o i s seconda y me aboli es o he ea men and p e en ion o demen ia and u he neu odegene a i e diseases [7,9-12]. Because o hei in e es ing biological ac i i ies and hei high po en ial in medicinal and pha maceu ical ap- plica ions, cya hane di e penoids ha e a ac ed inc easing in e es in ecen yea s. A p esen , he mos common me hod o analysis o cya hane di e penoids is high pe o mance liquid ch oma og aphy (HPLC) coupled o diode a ay and mass spec ome ic de ec ion [13]. As a apid, sensi i e and cos e ec i e me hod, an ELISA speci ic o cya hane ype seconda y ungal me ab- oli es may se e as a complemen a y me hod, especially in * Co espondence: [email p o ec ed] 1 Jus us Liebig Uni e si y Giessen, Ins i u e o Food Chemis y and Food Bio echnology, Hein ich-Bu -Ring 58, Giessen 35392, Ge many Full lis o au ho in o ma ion is a ailable a he end o he a icle © 2014 Shen e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly c edi ed. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Shen e al. BMC Bio echnology 2014, 14:98 h p://www.biomedcen al.com/1472-6750/14/98 sc eenings o new p oduce s ains. In he p esen s udy, an indi ec compe i i e ELISA o he s uc u e speci ic de ec ion o cya hane ype di e penoids using polyclonal an ibodies was de eloped, and i s po en ial o he analysis o biological samples was p o en. Resul s Op imum coa ing condi ions S ia al B was chosen as coa ing hap en in ELISA, because i is mo e pola han s ia al A. In o de o inc ease i s solubili y in aqueous solu ion, an equal olume o DMSO was added o he bu e s. The op imum coa ing condi ions (bes signal- o-noise a io and concen a ion dependence) we e ob ained by coa ing he pla e in PBS/DMSO (1/1, / ) bu e . Mo e in ense signals we e ob ained by coa ing a 24°C o 37°C, compa ed o coa ing a 4°C. S ia al B was adso bed mo e e icien ly by coa ing o e nigh han o 2 h. Based on hese esul s, he op imum coa ing condi ions o s ia al B a e summa ized as ollows: PBS/DMSO (1/1, / ) bu e , and coa ing o e - nigh a 24°C. The op imum concen a ion o coa ing o he hap en and he dilu ion o polyclonal an ibodies (pAbs) we e de e mined by checke boa d i a ion o be 5 μgmL −1 and 1:200, espec i ely, which we e used in he ollowing indi ec compe i i e ELISA expe imen s. Tole ance agains o ganic sol en s Due o he poo solubili y o cya hane di e penoids in aqueous solu ions, he ole ance agains o ganic sol en s used o dissol e hese compounds was es ed o assay op imiza ion. An addi ion o 5% DMSO, ace oni ile o me hanol, espec i ely, o he p e-incuba ion solu ion was ound accep able. Me hanol could be used up o 25% wi h- ou a no able nega i e e ec . High pe cen ages o DMSO up o 30% and o ace oni ile up o 20% esul ed in consid- e able nega i e e ec s on he assay. An addi ion o DMSO >30% o ace oni ile >20% showed a sudden inc ease o signals. Since me hanol eac s wi h s ia als [4], an addi ion o 5% DMSO o ace oni ile was selec ed o p e- incuba ion in he ollowing indi ec compe i i e ELISA. C oss eac i i y Figu e 2 illus a es a ypical s anda d calib a ion cu e o s ia al B. The speci ici y o he pAbs agains s ia al B was e alua ed by de e mina ion o he c oss eac i i ies o se e al s uc u ally ela ed compounds, such as s ia- als A, C, and D, s ia ins A and B, and e inacines C and P. The IC 50 alues a e gi en in Table 1. All o he es ed cya hane di e penoids showed c oss eac i i ies agains s ia al B. No colo eac ion was de ec ed wi h non- coa ed pla es as blanks. Assay p ecision In a-assay and in e -assay p ecisions we e de e mined o se e al concen a ions o s ia al B (Table 2). The in a-assay p ecision was calcula ed om he ex inc ion di e ence a 450 nm and 630 nm wi hin one mic o i e pla e. Less han 10% CV we e ob ained in e e y expe i- men , indica ing an accep able p ecision. The in e -assay p ecision was ob ained om %B/B 0 - alues o 8 pla es on di e en days. Va ia ions o less han 20% we e obse ed. The a e age IC 50 o s ia al B s anda d was 36.0 ng mL −1 wi h an in e -assay CV o 13.2% (n = 5). Reco e y To assess he assay accu acy, s ia al B was added o he sample ma ix in se e al concen a ions, and he eco - e ies o he spiked samples we e de e mined. In pa allel, HPLC-DAD was used o analyze he spiked samples. The appa en eco e ies o s ia al B in he sample ma ix as de e mined by indi ec compe i i e ELISA anged om 97.3% o 125.9%, while hose ob ained by HPLC-DAD a ied om 95.8% - 110.2%. The limi o quan i ica ion was 0.02 μgmL −1 o ELISA, and 0.40 μg OH O OO OR 2 H CHO H H R 1 S ia al A: R1= H, R2= COCH3 S ia al B: R1=OH, R2= COCH3 S ia al C: R1= OH, R2= H S ia al D: R1= R2= H OH O OO OR 2 HH H R 1 OH H 3 CO S ia in A: R1= H, R2= COCH3 S ia in B: R1=OH, R2= COCH3 S ia in C: R1= OH, R2= H O O HOOH OH OH E inacine C O O HOH OH OH H O O O E inacine P Figu e 1 S uc u es o s ia als, s ia ins and e inacines. Shen e al. BMC Bio echnology 2014, 14:98 Page 2 o 10 h p://www.biomedcen al.com/1472-6750/14/98 y = -0.722ln(x) - 2.3774 R = 0.9904 -4 -3 -2 -1 0 1 2 3 4 0.001 0.01 0.1 1 10 Logi (%B/B0) s ia al B [ g mL-1 ] calib a ion o s ia al B Log. (calib a ion o s ia al B ) a) b) Figu e 2 Calib a ion g aphs o he quan i ica ion o s ia al B. (a) Example o sigmoidal calib a ion cu e o quan i ica ion o s ia al B by indi ec compe i i e ELISA. (b) Example o linea i y o s ia al B calib a ion cu e. The linea wo king ange was ound o be 0.001 - 2.5 μgmL −1 , y=−0.722 ln(x) –2.3774, R 2 = 0.9904. Table 1 C oss eac i i ies o s uc u ally ela ed compounds o s ia al B Compounds C oss eac i i y [%]* S ia al B 100.0 S ia al A 39.8 S ia al C 77.8 S ia al D 74.4 S ia in A 24.1 S ia in B 109.3 E inacine C 60.0 E inacine P 21.1 *Rela ed o molecula a io. Table 2 In a- and in e -assay p ecision Concen a ion [μgmL −1 ] In a-assay (n = 3) CV [%] In e -assay (n = 8) CV [%] 0.63 2.7 16.3 0.32 3.9 14.8 0.16 1.7 16.3 0.08 2.4 8.3 0.04 4.5 10.4 0.0006 5.1 3.6 Shen e al. BMC Bio echnology 2014, 14:98 Page 3 o 10 h p://www.biomedcen al.com/1472-6750/14/98 mL −1 o HPLC-DAD. A good co ela ion be ween he s ia al B concen a ions measu ed by ELISA and by HPLC-DAD (y = 1.1122× –0.1585, R 2 = 0.9942) was ob- se ed (Figu e 3, Table 3). Analysis o H. e inaceus The indi ec compe i i e ELISA was applied o samples p epa ed om subme ged cul u es as well as om ui - ing bodies o H. e inaceus. The o al concen a ions o cya hane di e penoids in supe na an s and in ex ac s he eo a e displayed in Figu e 4. A simila change o % B/B 0 - alues was obse ed in bo h cases du ing he 8 cul u e days. %B/B 0 - alues in ex ac s o ui ing bodies om H. e inaceus we e nea o 100% and we e inde- penden om he sample concen a ion in he assay. Discussion In he p esen s udy, s ia al B was di ec ly a ached as a low molecula weigh hap en o mic o i e pla es. In many s udies he use o p o ein conjuga ed hap ens o immobiliza ion was epo ed [14-16], as he abili y o p o eins o adso b o he plas ic su ace o mic o i e pla es is much highe han ha o low molecula weigh hap ens. Howe e , he coupling a io o hap ens o ca - ie p o eins may a y depending on he coupling eac ion and a was e o aluable hap ens du ing conju- ga e syn hesis is o en una oidable. The in ol emen o ca ie p o eins esul s in se e al p oblems. Fi s ly, i may cause unspeci ic binding o pAbs and hus educe speci ic signals. Secondly, due o he la ge molecula dimension o ca ie p o eins, a shielding o masking e ec may occu ha impedes he binding o pAbs o he conjuga ed hap ens [17]. Some esea che s epo ed abou p e- ea men s o he pla e su ace, e.g. by UV i adia ion o eagen s in o de o ealize a di ec bind- ing o small molecules [18-20]. In he p esen s udy, he a ge analy e (molecula weigh <500 g mol −1 )was di ec ly a ached o mic o i e pla es wi hou p e- Figu e 3 Co ela ion be ween s ia al B concen a ions in spiked samples measu ed by ELISA (x-axis) and by HPLC-DAD (y-axis). Table 3 Reco e y o spiked s ia al B in sample ma ix Spiked concen a ion [μgmL −1 ] ELISA (n = 3) HPLC-DAD (n = 2) De ec ed [μgmL −1 ] Reco e y [%] De ec ed [μgmL −1 ] Reco e y [%] 10.010 ou o calib a ion - 10.50 ± 0.05 104.8% 5.005 ou o calib a ion - 5.26 ± 0.10 105.1% 2.002 1.949 ± 0.219 97.3% 2.03 ± 0.08 101.5% 1.001 1.048 ± 0.199 104.7% 0.96 ± 0.02 95.8% 0.501 0.630 ± 0.291 125.9% 0.50 ± 0.00 99.6% 0.400 0.478 ± 0.062 119.3% 0.44 ± 0.02 110.2% 0.200 0.252 ± 0.026 113.0% n.d. - 0.020 0.021 ± 0.001 102.8% n.d. - 0.001 n.d* - n.d. - *n.d: no de ec ed. Shen e al. BMC Bio echnology 2014, 14:98 Page 4 o 10 h p://www.biomedcen al.com/1472-6750/14/98 ea men o he pla e su ace. Signi ican di e ences be ween he a ious coa ing p ocedu es we e ound du ing he s udy o coa ing condi ions. The pH o he coa ing bu e signi ican ly a ec ed he coa ing e iciency. The inal pH o he coa ing bu e PBS, po assium ace a e and sodium ca bona e a e mixing wi h DMSO was 9.7, 5.8 and 11.1, espec i ely. An in luence o he pH on he coa ing e iciency has been epo ed in se e al publica ions [21-23]. In addi ion, a decomposi ion o s ia al B was de ec ed in po assium ace a e bu e in a ime cou se s udy by HPLC analysis (da a no shown). Tha migh be a eason o he poo a achmen o s ia al B o he pla e in his bu e sys em. Fo s ia al B, coa ing a pH 9.7 was mo e e icien han a pH 11.1. The p esence o o ganic sol en s may in luence he pe o mance o an ELISA. Me hanol caused he leas nega i e e ec , and he use o low concen a ions o ace o- ni ile o DMSO was also accep able. Simila esul s ha e been epo ed e.g. by [17,23,24]. The ole ance agains o ganic sol en s allows o he analysis o samples om ungal cul u es which equi e ex ac ion wi h an o ganic sol en . The sudden inc ease o he signals in he p esence o ace oni ile and DMSO in high concen a ions indica ed a alse posi i e signal possibly caused by dena u a ion o he an ibodies and hus an unspeci ic adso p ion o he blocking p o eins. All o he examined cya hane di e penoids showed c oss eac i i ies agains s ia al B. As expec ed, s ia in B wi h IC 50 = 109.3% showed he highes c oss eac i i y. S ia in B and s ia al B di e only in he aldehyde g oup a he C 7 ing, and his posi ion o s ia al B was used o BSA-conjuga e syn hesis o immuniza ion. The e o e, s ia in B and s ia al B possess he same an igenic de e minan s. E inacine P showed he lowes c oss eac i - i y wi h IC 50 = 21.1%. Based on he c oss eac i i ies and molecula s uc u es i migh be suspec ed ha he esul ing an ise um displays he highes a ini y owa d he cya hane ing moie y. The eco e y expe imen s sugges ed ha he de eloped ELISA is accu a e and signi ican ly mo e sensi i e han he HPLC-DAD me hod. I could be used o he es ima- ion o he o al cya hane di e penoid concen a ions o biological samples. The assay showed in e e ences wi h cons i uen s o he cul u e media o H. e inaceus,asa ma ix e ec was obse ed wi h he con ol samples (c . Figu e 4, con ol). The e o e, he use o o ganic ex ac s is p e e ed o he analysis o cya hane ype di e penoids in ungal cul u e supe na an s. The p oduc concen a ions could be es ima ed du ing he cul i a ion pe iod. How- e e , he o al concen a ions ep esen he sum o all compounds showing c oss eac i i y agains s ia al B. They may hus di e om he indi idual concen a- ions de e mined by HPLC. No signi ican in e e ences (%B/B 0 - alues close o 100%) we e obse ed wi h c ude ex ac s o ui ing bodies o H. e inaceus. The e o e, he seconda y me aboli es p esen in he ui ing bodies, e.g. he icenone and e inace ine, did no c oss eac wi h he polyclonal an ibodies. These esul s a e in good ag eemen wi h a e iew a icle abou seconda y me aboli es om H. e inaceus [12]. Up o da e, e inacines ha e been mos ly epo ed om subme ged cul u es o H. e inaceus, and only aces o e inacines ha e been ound in ui ing bodies [25]. In he p esen s udy, he ace concen- a ions o e inacines in he ui ing bodies we e appa en ly below he de ec ion limi o he ELISA. The CV% alues o he in e -assay p ecision migh be caused by many ac o s, e.g. he posi ion o he wells in he pla e (edge e ec ), he day o day a ia ion in eagen p epa a ion, he empe a u e o bu e s, he a iabili y in washing p ocedu es, pipe ing e o s and he loss o en- zyme ac i i y o he seconda y an ibodies a e p olonged s o age [16,26]. Conclusions Polyclonal an ibodies agains s ia al A/B we e p oduced, and an indi ec compe i i e ELISA was de eloped o 0 20 40 60 80 100 120 con ol day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8 day 9 % B/B 0 supe na an s ex ac s o supe na an s Figu e 4 Analysis o supe na an s o subme ged cul u es o H. e inaceus and ex ac s he eo du ing 8 cul u e days. Con ol: cul u e medium wi hou H. e inaceus. Shen e al. BMC Bio echnology 2014, 14:98 Page 5 o 10 h p://www.biomedcen al.com/1472-6750/14/98 de e mina ion o s ia al B and s uc u al ela ed com- pounds. The me hod has shown sa is ac o y esul s con- ce ning speci ici y, sensi i i y and accu acy. The applica ion o samples om subme ged cul u es and ui ing bodies o H. e inaceus indica ed ha he ELISA maybeused o hees ima iono o alcya hanedi e - penoid concen a ions in complex mix u es. Due o i s high sensi i i y and speci ici y, i ep esen s an ideal sc eening ool o disco e ing new po en ial p oduce s o cya hane ype di e penoids. Fu he op imiza ion will be needed o imp o e he in e -assay p ecision. Me hods Chemicals and solu ions All eagen s we e o analy ical g ade unless speci ied o he wise. Ul a-pu e wa e (p oduced by a ium® 611 VF Wa e Sys em, Sa o ius, Gö ingen, Ge many) was used o all solu ions, media, and HPLC eluen s. Ace oni ile (HPLC g adien g ade), calcium ca bona e, Edamin® K, 30% hyd ogen pe oxide (H 2 O 2 ), and D-manni ol we e pu chased om Sigma Ald ich (S einheim, Ge many). Sal s, Aga -Aga , bo ine se um albumin (BSA), EDTA, α-D(+)-glucose monohyd a e, soy pep one, suc ose, 3,3’,5,5’- e ame hyl benzidine (TMB), Tween® 20, yeas ex ac , and o ganic sol en s we e pu chased om Appli- chem (Da ms ad , Ge many) o om Ro h (Ka ls uhe, Ge many). Mal ex ac was ob ained om Fluka, Neu-Ulm, Ge many. Molasses was ob ained om Südzucke , O s ein, Ge many. Oa meal was p o ided om D . Oe ke , Düsseldo , Ge many. S anda ds o e inacines and s ia als, o iginally isola ed om he wo selec ed p oduce s ains, we e ob ained om he lib a y o pu e na u al p oduc s o In e Med Disco e y, Do mund, Ge many (IMD, o me ly Baye Heal hca e) and hei pu i y and iden i y was con i med by 2D-NMR and HR-MS, p io o hei use in he expe i- men s. Addi ionally, s ia als A and B ( o immuniza ion) and s ia als C and D, s ia ins A and B, and e inacines C and P ( o de e mina ion o c oss eac i i ies) we e ex ac ed and pu i ied om subme ged cul u es o he basidiomyce es Cya hus s ia us and He icium e ina- ceus. The s uc u es we e con i med by compa ison o he pu e e e ence compounds and by NMR analysis (c . Addi ional ile 1: Supplemen a y ma e ial). Solu ions o ELISA: Coa ing bu e s: 1. 10 mM phospha e bu e ed saline (PBS), pH 7.4 mixed wi h DMSO, 1/1, / . 2. 0.1 M po assium ace a e bu e , pH 4.0 mixed wi h DMSO, 1/1, / . 3. 0.1 M sodium ca bona e bu e , pH 9.6 mixed wi h DMSO, 1/1, / . Washing bu e : 10 mM PBS, pH 7.4 wi h 0.05% Tween® 20 (PBST). Blocking eagen (1% gela in) was pu chased om Roche Diagnos ics (Mannheim, Ge many). Seconda y an ibodies (goa an i- abbi lgG pe oxidase conjuga e) we e ob ained om Me ck (Da ms ad , Ge many). Colo de elopmen : 0.12 mg mL −1 TMB and 0.05% H 2 O 2 in 0.05 M sodium ace a e solu ion, pH 4.5 (TMB solu ion). This solu ion was eshly p epa ed be o e use. S opping eagen : 0.5 M sul u ic acid. Fungal s ains Cya hus s ia us (STMA07048, isola ed om basidio- spo es o a specimen om a unk o Picea collec ed in July 1997 in S elzenbe g, Rheinland-P alz, Ge many) and He icium e inaceus (FU70034, isola ed om basidioca p issue) o subme ged cul u e we e ob ained om IMD. P io o he expe imen s, he iden i y o bo h s ains was con i med by mic oscopic s udies and by compa ison o hei ITS n DNA sequences wi h e e ence da a in Genbank. The ungi we e main ained on a solid medium con aining 20 gL −1 mal ex ac and 15 g L −1 Aga -Aga .They a e deposi ed in he cul u e collec ion o IMD Na u al Solu ions GbR ( o me ly In e Med Disco e y GmbH unde liquid ni ogen. A duplica e s ain and he co esponding specimen o he C. s ia us ma e ial is also main ained a he pe sonal he ba ium and cul u e collec ion o Ma c S adle . F ui ing bodies o He icium e inaceus we e ob ained om a comme cial p o ide www.pilzga en.de, Hel esiek, Ge many. Cul u e media Soy pep one, yeas mal , and suga molasses media we e p epa ed acco ding o [25]. Ins umen s HPLC-DAD The HPLC sys em was om Me ck Hi achi, Da ms ad , Ge many, and comp ised a pump (L-7100), an au o sam- ple (L-7200), an in e ace (D-7000), and a diode a ay de ec o (L-7455, 200 –600 nm). Column: e e sed phase, Nucleosil® 100–5C 18 , CC 125/3 mm wi h a gua d column Nucleosil® 100–5C 18 , CC 8/3 mm (Mache ey Nagel, Dü en, Ge many). Flow a e: 0.4 mL min −1 o s ia al and s ia in analysis and 0.6 mL min −1 o e inacine analysis. Eluen : Ace oni ile (A) and ul a-pu e wa e (B). Wa eleng h: 210, 233 and 254 nm. G adien o s ia al and s ia in: 50% A (0 min) –94% A(14min)–94% A (17 min) –100% A (19 min) –100% A(25min)–50% A (30 min) –50% A (40 min). Shen e al. BMC Bio echnology 2014, 14:98 Page 6 o 10 h p://www.biomedcen al.com/1472-6750/14/98 G adien o e inacine: 30% A (0 min) –50% A (15 min) –50% A (16 min) –100% A (23 min) –100% A (38 min) –30% A (43 min) –30% A (47 min). P epa a i e HPLC A semi-p epa a i e HPLC sys em (Young Lin, Hongye Anyang, Ko ea) equipped wi h qua e na y pump (YL9110S) and dual wa eleng h UV/Vis de ec o (YL 9120S), com- bined wi h a ac ion collec o (CHF 122SC, Ad an ec, Osaka, Japan) was used. A column K omasil 100 C 18 ,7μm, 250 × 20 mm (MZ Analysen echnik, Mainz) wi h a gua d column K omasil 100 C 18 ,7μm, 50 × 20 mm (MZ) was used o isola e he a ge compounds. Flow a e: 15 mL min −1 . Eluen : Ace oni ile (A) and ul a-pu e wa e (B). G adien o s ia als and s ia ins: 50% A (0 min) – 50% A (10 min) –80% A (55 min) –80% A (70 min) – 100% A (80 min) –100% A (95 min). Wa eleng hs: 210 and 254 nm. G adien o e inacines: 25 A% (0 min) –25% A (10 min) –48% A (35 min) –48% A (45 min) –55% A (52 min) –55% A (60 min) –100% A (90 min) –100% A (110 min). Wa eleng hs: 210 and 233 nm. ELISA The ELISA was pe o med in mic o i e pla es Immuno Pla e Maxiso p F96 (Nunc, Denma k). Incuba ion s eps and colo de elopmen we e pe o med in an incuba o INE 500 (Memme , Schwabach, Ge many). Washing was done by an eigh -channel pipe e Resea ch P o 50–1200 μL (Eppendo , Wesseling-Be zdo , Ge many). The ex inc- ion was measu ed by a mic o i e pla e eade Syne gy 2 (BioTek, Bad F ied ichshall, Ge many). Biosyn hesis and isola ion o cya hane di e penoids Subme ged cul i a ion P e-cul u es o C. s ia us we e g own subme ged in 250 mL E lenmeye lasks con aining 100 mL soy pep one medium a 24°C and 150 pm o 7 days. A e ha , he mycelium was homogenized wi h an Ul a- Tu ax (IKA, S au en, Ge many) a 10,000 pm o 10 sec. 40 mL homogenized mycelium we e inocula ed in o a new 1,000 mL E lenmeye lask con aining 400 mL soy pep one medium and incuba ed a 24°C and 150 pm o u he 12 days. P e-cul u es o H. e inaceus we e g own subme ged in 250 mL E lenmeye lasks con aining 100 mL yeas mal medium a 24°C and 150 pm o 7 days. A e - wa ds, 40 mL homogenized mycelium we e inocula ed in o 400 mL suga molasses medium in 1,000 mL E lenmeye lasks and incuba ed a 24°C and 150 pm o u he 4 days o ob ain e inacine P o 12 days o ob ain e inacine C. Ex ac ion and isola ion A e subme ged cul i a ion, he mycelia om 2.2 L cul u e media we e sepa a ed om he supe na an s by cen i- uga ion a 2,880 gand 4°C o 10 min (Alleg a® X-15R, BECKMAN COULTER™,K e eld,Ge many),andex- ac ed wice wi h 400 mL e hyl ace a e. The combined o ganic ex ac s we e d ied o e sodium sul a e, and a e wa ds e apo a ed o d yness. A b own esidue was ob ained a e e apo a ion, which was dissol ed in ace oni ile and subjec ed o p epa a i e HPLC. The ex- ac s o Cya hus s ia us con ained he s ia als A, B, C and D. The s ia ins A and B we e p epa ed by con e sion o he s ia als A and B by s i ing in me hanol a oom empe a u e o e nigh , espec i ely. The e inacines P and C we e isola ed om he ex ac s o He icium e inaceus. P oduc ion o polyclonal an ibodies The aldehyde unc ion o he s ia als and NH 2 unc ions o BSA we e used o syn hesis o s ia al A/B (1/1, w/ w)-BSA conjuga es. The a io o s ia als o BSA was de- e mined by MALDI-TOF-MS. Abou 17 s ia al mole- cules we e coupled o one BSA molecule (Eu ogen ec S. A, Se aing, Belgien). The p oduc ion o abbi pAbs agains s ia al A/B was pe o med by Eu ogen ec S.A. Two abbi s wi h ID SA6928 and SA6929 we e immu- nized ou imes wi h 200 μg o he s ia al A/B-BSA conjuga e on day 0, 14, 28 and 56. An addi ional immuniza ion was done on day 91wi h 400 μg s ia al A/B-BSA conjuga e. The inal an ise a we e collec ed on day 115 and s o ed a −20°C wi hou u he pu i ica ion un il use. The p e-bleed se a o hese wo abbi s on day 0 be o e immuniza ion we e used as nega i e con ol in ELISA. The inal an ise um om abbi SA6928 was used in he ollowing assays because o i s highe speci ici y agains s ia al B compa ed o SA6929 (da a no shown). De elopmen o an indi ec compe i i e ELISA Op imiza ion o coa ing condi ions S ia al B was di ec ly a ached o he pla e in se e al di e en concen a ions. The op imal condi ions (bu e , empe a u e and ime) o di ec coa ing o s ia al B we e s udied by an indi ec non-compe i i e ELISA. The incuba ion empe a u es we e 4°C, 24°C and 37°C, and he coa ing ime was 2 h o o e nigh . Checke boa d i a ion The op imal dilu ion o pAbs and he op imal coa ing amoun o s ia al B we e de e mined simul aneously by checke boa d i a ion (a wo-dimensional i a ion me hod) by indi ec non-compe i i e ELISA. S ia al B wi h di e en concen a ions (0.04 o 20 μgmL −1 , wo old dilu ion) was coa ed o he mic o i e pla e and hen bound o pAbs wi h se ial dilu ions (1:50 o 1:6,400, s epwise in wo old dilu ion). Shen e al. BMC Bio echnology 2014, 14:98 Page 7 o 10 h p://www.biomedcen al.com/1472-6750/14/98 Tole ance agains o ganic sol en s The ole ance agains o ganic sol en s (DMSO, ace oni ile, me hanol) used o dissol e he cya hane di e penoids was examined by indi ec compe i i e ELISA. Di e en p opo - ions o o ganic sol en (5 - 50%, / , 0% as blank) we e added o PBST wi h a inal dilu ion o pAbs 1:200 and p e-incuba ed wi hou compe ing molecules a 24°C o 1 h. The e ec s o o ganic sol en s on he ELISA sys em we e e alua ed by compa ing he measu ed ex inc ions o hose o he blanks. ELISA Coa ing 100 μLo 5μgmL −1 s ia al B in coa ing bu e 1 we e pi- pe ed in wells o a mic o i e pla e. 100 μLo 1μgmL −1 BSA dilu ed in PBS we e pipe ed in wo wells as posi i e con ol. The incuba ion was pe o med o e nigh a 24°C. Blocking 200 μL o 1% gela in we e added o he wells and incu- ba ed a 24°C o 3 h. P e-incuba ion Se ial dilu ions o analy e we e p epa ed in a concen a- ion ange o 0.0003 - 20 μgmL −1 in PBST con aining 5% ace oni ile o DMSO and a inal pAbs dilu ion o 1:200. The incuba ion was pe o med o 1 h a 24°C in 2 mL Eppendo ® cups. Incuba ion wi h pAbs 100 μL p e-incuba ed analy e we e added o each well (in iplica e) and incuba ed o 1 h a 24°C. Fo he indi ec non-compe i i e ELISA, 100 μL pAbs wi h a 1:100 dilu ion in PBST we e used wi hou compe i o s ins ead o he p e-incuba ed analy e. Incuba ion wi h seconda y an ibodies 100 μL seconda y an ibodies wi h a 1:5,000 dilu ion in PBST we e added o each well and incuba ed o 1 h a 24°C. Colo de elopmen 100 μL o he TMB solu ion we e added o each well and incuba ed o 15 min a 24°C. Wi hou washing s ep, 100 μL o 0.5 M sul u ic acid we e added o each well o s op he colo de elopmen . Measu emen The ex inc ions we e measu ed immedia ely by a mic o- i e pla e eade a 450 nm and 630 nm. Unless o he wise speci ied, he wells we e washed wi h 300 μL washing bu e PBST h ee imes a e each incu- ba ion s ep. The indi ec non-compe i i e ELISA in ol ed all o he s eps o he indi ec compe i i e ELISA, excep o he p e-incuba ion o pAbs wi h compe i o s. Da a analysis S ia al B s anda ds and samples we e analyzed in ipli- ca es. The di e ences be ween ex inc ions a 450 nm and 630 nm we e used o calcula ion and plo ing he sigmoidal cu es. %B/B 0 - alues o calib a ion cu es we e calcula ed as ollows: %B B0 ¼ΔE−ΔEexcess ΔE0−ΔEexcess 100 ΔE: he ex inc ion di e ences o samples a 450 nm and 630 nm. ΔE 0 : he ex inc ion di e ences o he uppe asymp o e o he sigmoidal cu e be ween 450 nm and 630 nm. ΔE excess : he ex inc ion di e ences o he lowe asymp- o e o he sigmoidal cu e be ween 450 nm and 630 nm. C oss eac i i y (IC 50 ) The assay speci ici y was e alua ed by ob aining sig- moidal cu es o se e al s uc u ally ela ed compounds (s ia als A, C and D, s ia ins A and B, and e inacines C and P) as compe i o s agains he s ia al B s anda d. The calib a ion cu e o s ia al B s anda d was mea- su ed indi idually o each pla e. The IC 50 we e es i- ma ed by using a linea iza ion o he calib a ion cu es wi h a logi - ans o ma ion o %B/B 0 - alues: logi %B=B0 ðÞ¼ln %B=B0 100−%B=B0  C oss eac i i y (IC 50 ) = A/B × 100%. A: concen a ion o s ia al B s anda d a logi (%B/B 0 )=0. B: concen a ion o compe i o s a logi (%B/B 0 ) =0. The linea eg ession o logi (%B/B 0 ) was used o de- e mina ion o concen a ions in unknown samples. Reco e y Ace oni ile was spiked wi h s ia al B a se e al concen- a ions (0.001 - 10 μgmL −1 ) and analyzed by indi ec compe i i e ELISA as well as by HPLC-DAD. The concen- a ions o s ia al B o spiked samples we e calcula ed om linea eg ession o logi (%B/B 0 ) in ELISA and linea calib a ion o s ia al B in HPLC-DAD. The eco e ies o s ia al B we e ob ained by: eco e y %½¼ de ec ed concen a ion spiked concen a ion 100% Analysis o H. e inaceus samples The supe na an s and ex ac s o supe na an s om sub- me ged cul u es, as well as ex ac s o ui ing bodies o H. e inaceus we e analyzed by indi ec compe i i e ELISA: Shen e al. BMC Bio echnology 2014, 14:98 Page 8 o 10 h p://www.biomedcen al.com/1472-6750/14/98 Sample p epa a ion o p e-incuba ion o supe na an s in ELISA 50 μL o supe na an s om subme ged cul u es (cul u e day 1 o day 8) we e added o 950 μL PBST wi h a inal DMSO p opo ion o 5% and a inal pAbs dilu ion o 1:200. Cul u e medium wi hou H. e inaceus was used as nega i e con ol. Sample p epa a ion o p e-incuba ion o ex ac s o supe na an s 20 mL o supe na an s om subme ged cul u es (cul u e day 1 o 8) we e ex ac ed once wi h 20 mL e hyl ace - a e. 15 mL o he e hyl ace a e phase was e apo a ed o d yness, and he esidues we e edissol ed in 1 mL ace oni ile o ELISA and HPLC-DAD analysis. 5 μLo he ex ac s we e added o 995 μL PBST wi h a inal ace oni ile p opo ion o 5% and a inal pAbs dilu ion o 1:200. Ex ac s o cul u e media wi hou H. e inaceus we e used as nega i e con ols. Sample p epa a ion o p e-incuba ion o ex ac s o ui ing bodies 32.6 g ui ing bodies o H. e inaceus we e homogenized and hen ex ac ed wi h 200 mL e hyl ace a e unde s i - ing o e nigh . The e hyl ace a e phase was d ied o e sodium sul a e and e apo a ed o d yness a e il a ion. 1 mg c ude ex ac was dissol ed in 1 mL ace oni ile. Se e al concen a ion le els (50.0, 10.0, 5.0, 2.5, 1.0 and 0.5 μgmL −1 ) o he ex ac s we e used in he p e- incuba ion solu ion. Ace oni ile was used as blank. A ailabili y o suppo ing da a The da a se s suppo ing he esul s o his a icle a e included wi hin he a icle and i s addi ional ile. Addi ional ile Addi ional ile 1: Supplemen a y ma e ial. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Au ho s’con ibu ions TS pu i ied he cya hane ype di e penoids, ca ied ou he ELISA and d a ed he manusc ip . LH was in ol ed in he s udies on he c oss eac i i ies, and HH was esponsible o NMR analyses and da a in e p e a ion. MS p o ided he ungal s ains and con ibu ed o he discussion. HZ pa icipa ed in he design o he s udy and helped o d a he manusc ip . All au ho s ead and app o ed he inal manusc ip . Acknowledgemen The au ho s hank he BMBF and CLIB 2021 o inancial suppo o he p ojec (FKZ 0315404 A). Pa o he wo k was suppo ed by he excellence ini ia i e o he Hessian Minis y o Science and A which encompasses a gene ous g an o he LOEWE esea ch ocus ‘In eg a i e Fungal Resea ch’. Au ho de ails 1 Jus us Liebig Uni e si y Giessen, Ins i u e o Food Chemis y and Food Bio echnology, Hein ich-Bu -Ring 58, Giessen 35392, Ge many. 2 Jus us Liebig Uni e si y Giessen, Ins i u e o O ganic Chemis y, Hein ich-Bu -Ring 58, Giessen 35392, Ge many. 3 Depa men Mic obial D ugs, Helmhol z Cen e o In ec ion Resea ch, Inho ens asse 7, B aunschweig 38124, Ge many. Recei ed: 2 June 2014 Accep ed: 5 No embe 2014 Re e ences 1. 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