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
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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
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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.
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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
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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.
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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.
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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).
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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
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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
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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
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