Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development

De ec ion o Aspe gillus umiga us myco oxins: immunogen syn hesis and immunoassay de elopmen M. Fox, G. G ay, K. Ka anagh, C. Lewis, S. Doyle* Na ional Ins i u e o Cellula Bio echnology, Depa men o Biology, Na ional Uni e si y o I eland Maynoo h, Maynoo h, Co. Kilda e, I eland Recei ed 25 Augus 2003; ecei ed in e ised o m 21 Oc obe 2003; accep ed 23 Oc obe 2003 Abs ac Immunological de ec ion o sec e ed low molecula weigh oxins ep esen s a po en ially no el means o diagnosing in ec ion by he ungus Aspe gillus umiga us. Two such me aboli es, glio oxin and hel olic acid, we e selec ed and conjuga ed o hy oglobulin o an ise a gene a ion in abbi s. Glio oxin was ini ially ac i a ed using N-[ p-maleimidophenyl] isocyana e (PMPI) and subsequen ly conjuga ed o S-ace yl hioglycolic acid N-hyd oxysuccinimide-ac i a ed hy oglobulin, whe eas hel olic acid was ac i a ed wi h N-(3-Dime hylaminop opyl)-NV-e hylca bodiimide (EDC) in he p esence o hy oglobulin p io o immunisa ion. To acili a e subsequen an ise a e alua ion, bo h oxins we e simila ly conjuga ed o bo ine se um albumin (BSA). Ma ix-Assis ed Lase Deso p ion Ionisa ion-Time O Fligh (MALDI-TOF) mass spec ome y and SDS- PAGE analysis con i med co alen a achmen o oxins o BSA in he a ios o 15 and 2.4 mol pe mol BSA o glio oxin and hel olic acid, espec i ely. Resul an high i e an ise a we e capable o de ec ing bo h BSA-conjuga ed oxins (inhibi o y concen a ion (IC) 50 : 4–5 Ag/ml). F ee oxins we e also de ec able by compe i i e immunoassay, whe eby 10 Ag/ml ee glio oxin (30 AM) and hel olic acid (17 AM), espec i ely, inhibi ed an ibody binding o cogna e oxin–BSA p e iously immobilised on mic owells. This wo k con i ms ha sensi i e and speci ic an ise a can be aised agains ungal oxins and may ha e an applica ion in diagnosing ungal in ec ion. D2003 Else ie B.V. All igh s ese ed. Keywo ds: Glio oxin; ELISA; MALDI-TOF; Hap en; Immunocomp omised 1. In oduc ion The ilamen ous ungus Aspe gillus umiga us is esponsible o a ange o pulmona y in ec ions in immunocomp omised pa ien s and hose wi h p e- exis ing lung damage (F ase , 1993; Daly and Ka anagh, 2001). In asi e aspe gillosis is he mos se ious o m o aspe gillosis, has a mo ali y a e o 80–95% and occu s almos exclusi ely in indi id- uals wi h p e-exis ing lung damage o disease and in hose immunocomp omised as a esul o disease o he apy (Denning, 1998). In his o m o he disease, he e is in asion and nec osis o he lung wall, in addi ion o whole body ungal dissemina- ion, which esul s in he in ec ion o a wide ange o o gans (Daly and Ka anagh, 2001).Despi e agg essi e an i- ungal chemo he apy, dea h usually esul s 7–14 days pos -diagnosis (Denning, 1996). As pa o i s complemen o i ulence a ibu es A. 0167-7012/$ - see on ma e D2003 Else ie B.V. All igh s ese ed. doi:10.1016/j.mime .2003.10.009 * Co esponding au ho . Tel.: +353-1-7083858; ax: +353-1- 7083845. E-mail add ess: [email protected] (S. Doyle). www.else ie .com/loca e/jmicme h Jou nal o Mic obiological Me hods 56 (2004) 221–230 umiga us p oduces a ange o oxins, mos p e- dominan ly he immunosupp essi e glio oxin (Ami- ani e al., 1995a,b), and enzymes (p o eases, elas ases, phospholipases) which hinde he hos immune esponse and acili a e issue pene a ion, espec i ely (Rinaldi, 1983). Fu he mo e, ex ac s ob ained om aspe gillosis pa ien spu um ha e been shown o damage human espi a o y epi helial cells (Ami ani e al., 1995a). Subsequen analysis con i med ha glio oxin de i ed om clinical iso- la es o A. umiga us was he oxic agen and ha hel olic acid also caused comple e cilios asis and epi helial cell dis up ion (Ami ani e al., 1995b). Cu en immunological es s o assess he p esence o aspe gillosis p ima ily ely upon he de ec ion o Aspe gillus an igens associa ed wi h he ungal cell wall (mannans o galac omannans), howe e , sub- op imal sensi i i y and speci ici y ha e esul ed in limi ed clinical applica ion (Meunie , 1996; Richa d- son and Kokki, 1998).Weig e al. (2001a,b) ha e p oposed he use o an i-mi ogillin (Asp 1) an ibody as an al e na i e me hod o he de ec ion o aspe gil- losis and, al hough con o e sial (Woo e al., 2001), his me hod may p o e o ha e a use ul clinical applica ion. Mo e ecen ly, Woo e al. (2002) ha e de eloped ELISA sys ems which de ec A. umiga us galac omannan (A mp1p) and an i-A mp1p an ibody in in asi e aspe gillosis pa ien s esul ing in a com- bined sensi i i y o 86.7%. Al hough he applica ion o Aspe gillus DNA de ec ion sys ems has p o en use ul in e ms o co ela ion in ungal DNA educ ion wi h disease esolu ion and ea men e icacy, he inabili y o nucleic acid-based sys ems o di e en ia e be ween (i) ungal s ains and (ii) colonisa ion and in ec ion emains p oblema ical (Yeo and Wong, 2002). Ob iously, he e is signi ican unde -de ec ion o aspe gillosis and in an e o o o e come his p oblem, no el me hods o he de ec ion o glio- oxin (e.g., RP-HPLC and in i o cell-based sys- ems which mimic he in e ac ion o ungal hyphae wi h human issue) ha e been de eloped (Belka- cemi e al., 1999; Daly and Ka anagh, 2002). Al hough hese sys ems ha e excellen po en ial o con i ma ion o Aspe gillus in ec ion hey a e no amenable o ou ine use in diagnos ic labo a o- ies. De ec ion o speci ic ungal me aboli es has been discussed as an al e na i e o an ibody, an igen o nucleic acid-based es s (Yeo and Wong, 2002). Glio oxin, a well-cha ac e ised ungal me aboli e, has po en immunosupp essi e e ec s and is indic- a i e o in asi e aspe gillosis (Denning, 1998). Simila ly, Mi chell e al. (1997) ha e shown ha hel olic acid, p oduced by A. umiga us, inhibi s he oxida i e bu s o mac ophages. Consequen ly, he appea ance o hese oxins may be indica i e o in asi e aspe gillosis since nei he oxin has been associa ed wi h in ec ions caused by o he clinically ele an ungi. Thus, de elopmen o immunoas- says, which could de ec one o mo e oxins, p oduced by he genus Aspe gillus, would ep esen a mo e eliable means o iden i ying unde lying ungal in ec ion and acili a e an e ec i e ea men p o ocol. The aims o he wo k p esen ed he e we e o de e mine i an ibodies could be p oduced agains ungal me aboli es and o assess he unc- ionali y o any esul an an ibodies by enzyme immunoassay. 2. Ma e ials and me hods 2.1. Chemicals All ma e ials we e pu chased om Sigma- Ald ich Chemical (Do se , UK) unless o he wise s a ed. 2.2. P epa a ion o hel olic acid–p o ein conjuga es Hel olic acid was p epa ed a 1 mg/ml in dime hyl sul oxide (DMSO). B ie ly, 100 AlN-(3-Dime hyla- minop opyl)-NV-e hylca bodiimide (EDC; 10 mg/ml in deionised wa e ) was added o 25 Al o he oxin o acili a e ca boxyl g oup ac i a ion (Fig. 1), ep e- sen ing a 100- old mola excess o EDC o e oxin. Immedia ely, 100 Al o ei he bo ine se um albumin (BSA) o hy oglobulin (10 mg/ml in 100 mM Me h- anesulphonic acid, 500 mM NaCl pH 6.0) was added o he hel olic acid/EDC mix u e, agi a ed gen ly and he eac ion allowed o p oceed o 2 h a oom empe a u e. This combina ion o eac an s ep e- sen ed a 3- and 0.3- old mola a io o ac i a ed hel olic acid o BSA and hy oglobulin, espec i ely. Addi ion o hyd oxylamine (10 mM inal concen a- ion) e mina ed he eac ion. The esul an conjuga es M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230222 we e dialysed wice, o 4 h on each occasion wi h s i ing, a 4 jC agains ei he phospha e bu e ed saline (PBS) (p io o immunisa ion and subsequen ELISA analysis) o deionised H 2 Op io omass spec ome y. 2.3. P epa a ion o glio oxin–BSA conjuga es S-ace yl hioglycolic acid N-hyd oxysuccinimide (SATA; 1 mg/50 Al dime hyl o mamide (DMF)) was added o 10 ml o BSA (1 mg/ml in 50 mM po assium phospha e, 150 mM NaCl, 1 mM EDTA (Bu e A), pH 7.8). The solu ion was mixed gen ly, allowed o incuba e o 1 h and dialysed ex ensi ely agains Bu e A a pH 6.8. Thy oglob- ulin was simila ly ac i a ed wi h SATA. The ex en o SATA inco po a ion was measu ed, and eac i e sulphyd al g oups exposed (deblocked), as p e i- ously desc ibed (Duncan e al., 1983). Glio oxin (2 mg/ml in DMSO) was ac i a ed h ough a ailable hyd oxyl g oups (Fig. 1) by adding 50 o 105 AlN- [p-maleimidophenyl] isocyana e (PMPI; 30 mg/ml in DMSO) (Annunzia o e al., 1993) and b ough o a inal olume o 310 Alwi hDMSOwhich ep esen ed a 5- old mola excess o PMPI o e glio oxin. A e eac ing o 1 h a oom empe a- u e, PMPI-ac i a ed glio oxin (260 Al) was added o 2 ml deblocked SATA-BSA o SATA- hy oglob- ulin (0.5 mg/ml in Bu e A pH 6.8). This combi- na ion o eac an s ep esen ed a 20- and 2- old mola excess o ac i a ed glio oxin o BSA and hy oglobulin, espec i ely. A e 2 h incuba ion, conjuga es we e dialysed as desc ibed abo e (Sec- ion 2.2). Bo h oxin–p o ein conjuga es we e ana- lysed by SDS-PAGE, Wes e n blo ing and Ma ix- Assis ed Lase Deso p ion Ionisa ion-Time O Fligh (MALDI-TOF) mass spec ome y. 2.4. Polyclonal an ibody gene a ion Fou abbi s (New Zealand Whi e emale abbi s. Age ange: 6–12 mon hs. Weigh ange: 3–4 kg. Ob ained om Ha lan UK, Oxon, UK) we e immu- nised in o al, wo wi h each oxin– hy oglobulin conjuga e. Animals we e ini ially immunised subcu- aneously wi h 50 Ag o each hy oglobulin– oxin conjuga e in F eunds comple e adju an ( inal ol- ume = 3 ml (50:50 F eunds comple e adju an : conju- ga e in PBS)). Fo subsequen immunisa ions ( ou , a wo-weekly in e als) 50 Ag o each hy oglobulin– oxin conjuga e in F eunds incomple e adju an (50:50 wi h conjuga e in PBS) was used un il a sa is ac o y i e (an igen de ec ion (1 Ag) by Wes e n blo a g ea e han o equal o 1/2000 an ise a dilu ion) was achie ed. Immunisa ions we e ca ied ou acco ding o I ish Depa men o Heal h Licence B100/2622. 2.5. P o ein– oxin conjuga e analysis F ee and modi ied oxin–BSA conjuga es we e e alua ed by SDS-PAGE and Wes e n blo analysis (Ennis e al., 2001). B ie ly, elec o ans e ed p o- eins we e de ec ed using dilu ed immune an ise um Fig. 1. S uc u es o glio oxin and hel olic acid used o conjuga ion o hy oglobulin and bo ine se um albumin (BSA), espec i ely. Glio oxin con ains wo hyd oxyl g oups a ailable o ac i a ion using N-[ p-maleimidophenyl] isocyana e (PMPI). Hel olic acid con ains a single ca boxyl g oup which can be ac i a ed using N-(3- Dime hylaminop opyl)-NV-e hylca bodiimide (EDC) p io o p o ein conjuga ion. M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230 223 Fig. 2. (A) SDS-PAGE analysis o oxin–BSA conjuga es (1 Ag/lane). Lane 1, molecula weigh ma ke s; Lane 2, BSA; Lane 3, hel olic acid– BSA and Lane 4, glio oxin–BSA. (B) Wes e n blo analysis o oxin–BSA conjuga es using abbi an ise a (1/2000). Immunoblo ing was ca ied ou in wo s ages, whe eby glio oxin–BSA conjuga e was ini ially de ec ed when an i glio oxin– hy oglobulin an ise a was added o he ni ocellulose memb ane. Subsequen memb ane incuba ion wi h an i hel olic acid– hy oglobulin an ise a e ealed he p esence o hel olic acid–BSA conjuga e. Lane 1, BSA; Lane 2, Hel olic acid–BSA and Lane 3, glio oxin–BSA. (C) MALDI-TOF mass spec ome y analysis o BSA, glio oxin–BSA and hel olic acid–BSA conjuga es. M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230224 (1/2000) in PBST/1%(w/ ) milk powde (Bu e B). Signal e ela ion was by goa IgG [an i- abbi IgG]– HRP conjuga e and he Diaminobenzidine/H 2 O 2 sub- s a e sys em. Mass spec ome y was ca ied ou using a B uke Bi lex 1V MALDI-TOF Mass Spec- ome e . All samples we e eeze-d ied o 1 mg/ml, deposi ed (1 Al) wi h 1 Al sinnipinic acid ma ix on o a mass spec ome y slide and allowed o d y p io o analysis. 2.6. ELISA p o ocols Toxin–BSA conjuga es we e indi idually dilu ed in 200 mM sodium ca bona e pH 9.6. Fla -bo omed mic o i e pla es (MaxiSo pkNunc-ImmunokMod- ules; Nalge NUNC In e na ional, Roskilde, Denma k) we e subsequen ly coa ed a 37 jC o 1 h wi h sa u a ing amoun s o hel olic acid–BSA conjuga es (1 Ag/ml; 100 Al/well). Op imal glio oxin–BSA coa - ing was ound o be 5 Ag/ml (100 Al/well). A e coa ing, mic o i e pla es we e washed wice wi h phospha e bu e ed saline–0.05%( / ) Tween-20 (PBST) ollowed by addi ion o blocking solu ion (1%(w/ ) BSA in coa ing bu e ; 200 Al/well) o s abilise bound oxin–p o ein conjuga es and mini- mise non-speci ic binding. Immune an ise a, dilu ed om 1/5000–1/40000 in PBST, was added wi hou he ele an oxin ( inal olume: 100 Al/well). In he case o compe i i e ELISA o ma s o bo h oxins, mic opla e coa ing a 5 Ag/ml was used o glio oxin Fig. 3. De e mina ion o an ise a eac i i y agains oxin–BSA conjuga es by ELISA. Fou abbi s we e immunised in o al, wo wi h each oxin– hy oglobulin conjuga e. (A) An ise a eac i i y agains immobilised glio oxin–BSA (02-5G and 02-6G) and (B) an ise a eac i i y agains immobilised hel olic acid–BSA (02-3H and 02-4H). Glio oxin– hy oglobulin an ise a e eals ela i ely high speci ici y o immobilised glio oxin BSA. Hel olic acid– hy oglobulin an ise a did no bind o immobilised glio oxin–BSA. Duplica e analysis was ca ied ou in all cases. M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230 225 and 0.05 Ag/ml o hel olic acid de ec ion, espec i e- ly. In addi ion, ele an an ise a and oxin we e ei he added immedia ely o p e-incuba ed o 1 o 16 h p io o addi ion o he mic o i e pla e. The ee oxin concen a ion ange was 0–50 Ag/ml. Following incu- ba ion (1 h), he pla e was washed ou imes wi h PBST and goa IgG [an i- abbi IgG]–HRP conjuga e added (100 Al/well; 1/1000 in Bu e B) o 1 h. The pla e was washed ou imes and e ame hylbenzidine subs a e (BioFX Labo a o ies, MD, USA) was added o 10 min. The eac ion was s opped by adding 1 N H 2 SO 4 and he abso bance was ead a 450/630 nm using an MRX mic o i e pla e eade (Dynex Tech- nologies, Wes Sussex, UK). 3. Resul s 3.1. Toxin–p o ein conjuga e o ma ion Toxin–p o ein conjuga es we e syn hesised (i) o enhance he immune esponse agains he oxin moi- e ies and (ii) o acili a e oxin immobilisa ion on mic opla es o enzyme immunoassay. Glio oxin- and hel olic acid–BSA conjuga es we e each analysed by SDS-PAGE, Wes e n blo and MALDI-TOF mass spec ome y (Fig. 2A–C). Wes e n blo analysis (Fig. 2B) was ca ied ou in wo s ages and only glio oxin–BSA conjuga e (lane 3, Fig. 2B)was de ec ed when an i glio oxin– hy oglobulin an ise a was added o he ni ocellulose memb ane and de ec ed as desc ibed in Sec ion 2.5. Subsequen memb ane incuba ion wi h an i hel olic acid– hy o- globulin an ise a e ealed he p esence o hel olic acid–BSA conjuga e. Unconjuga ed BSA was no de ec ed by any an ise um used and high molecula weigh conjuga e o ma ion is e iden in Fig. 2B (lanes 2–3), possibly as a esul o p o ein c oss-linking. SDS-PAGE and Wes e n blo analysis o glio oxin– BSA (Fig. 2A (lane 4) and B) esul ed in he de ec ion o a band a an appa en M o 78 kDa, which con i ms a loading o 15 mol glio oxin/mol BSA. In e es ingly, MALDI-TOF analysis o glio oxin–BSA indica es a hap en loading o only 4 mol glio oxin/mol BSA (Fig. 2C) (see Discussion). Al hough SDS-PAGE did no exhibi su icien esolu ion o de ec bound hel olic acid, MALDI-TOF analysis o he hel olic acid–BSA conjuga e con i med a hap en loading o 2.4 mol hel olic acid/mol BSA. Due o he ela i ely la ge size, hy oglobulin conjuga es could no be analysed by mass spec ome y and he deg ee o hap en loading could no be de e mined by SDS-PAGE due o limi- a ions in esolu ion and he la ge p o ein size ela i e o ha o he hap enyla ed o m. 3.2. An i- oxin an ise a i e and speci ici y e alua ion by ELISA Resul an an ibody i e s and speci ici y o all bo h immunogens is shown in Fig. 3.Fig. 3A illus a es he eac i i y o an ise a aised agains glio oxin- and hel olic acid– hy oglobulin conju- ga es, espec i ely, agains immobilised glio oxin– BSA o e a ange o an ise a dilu ions anging om 1/5000 o 1/40000. Speci ic de ec ion o immobilised glio oxin–BSA is e iden a an an i- se um (an i glio oxin– hy oglobulin) dilu ion o 1/ 40000 which was in u n selec ed as he op imal an ise um dilu ion o he de ec ion o ee glio- Fig. 4. Inhibi ion p o iles o oxin– hy oglobulin an ise a binding o immobilised oxin–BSA ollowing co-incuba ion wi h cogna e oxin–BSA conjuga e (-y-) ee BSA (-n-) o hy oglobulin (-E-). IC 50 alues anged om 4 o 5 Ag/ml. Duplica e analysis was ca ied ou in all cases. M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230226 oxin. Al hough some eac i i y is e iden a lowe dilu ions o hel olic acid– hy oglobulin an ise a, minimal c oss- eac i i y wi h bound conjuga e is de ec ed a an ise a le els g ea e han 1/10000 dilu ion. The eac i i y o an ise a agains immobilised hel olic acid–BSA conjuga e is shown in Fig. 3B. Signi ican ly, glio oxin– hy oglobulin an ise a does no bind o hel olic acid–BSA an igen a any dilu ion es ed. In all cases, p e-immune an ise a did no de ec ei he immobilised glio oxin- o hel olic acid–BSA conjuga es. Fu he mo e, immune an ise a did no bind o unmodi ied BSA immobilised a he same coa ing concen a ion as oxin–BSA conjuga es, he eby con i ming he speci ici y o he espec i e an ise a o he conjuga ed oxin moie ies. Fig. 5. (A) Inhibi ion o glio oxin– hy oglobulin an ise a (1/40000) binding o immobilised glio oxin–BSA (mic owell coa ing concen a ion: 5Ag/ml) by ee glio oxin. An ise a and ee glio oxin we e ei he added immedia ely o mic owells (-E-) o incuba ed o 1 h (-n-) o 16 h (-y-) p io o addi ion o coa ed mic owells. (B) Inhibi ion (41%) o glio oxin– hy oglobulin an ise a (1/5000) binding o immobilised glio oxin–BSA (mic owell coa ing concen a ion: 1 Ag/ml) by ee glio oxin. An ise a and ee glio oxin we e added immedia ely o mic owells a e mixing. (C) Inhibi ion o hel olic acid – hy oglobulin an ise a (1/2000 (-E-), 1/8000 (-n-) o 1/32000 (-y-)) binding o immobilised hel olic acid– BSA (mic owell coa ing concen a ion: 0.05 Ag/ml) by ee hel olic acid. An ise a and ee hel olic acid we e added immedia ely o mic owells and no p e-incuba ion was necessa y. Duplica e analysis was ca ied ou in all cases. M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230 227 3.3. Toxin–p o ein conjuga e inhibi ion o an ibody binding Fu he e alua ion o oxin–p o ein an ise a was pe o med by de e mining he ex en o inhibi ion o an ise a binding due o he p esence o ei he he app op ia e oxin–BSA conjuga e o ee BSA. I is clea om Fig. 4 ha ee BSA (o hy oglobulin) does no in e e e wi h an ibody binding o he e- spec i e immobilised oxin conjuga e. Howe e , he addi ion o ee oxin–BSA o glio oxin- and hel olic acid– hy oglobulin an ise a, espec i ely, a dilu ions o 1/40000 and 1/16000, comple ely inhibi s an ibody binding o he immobilised oxins wi h inhibi o y concen a ion (IC) 50 alues anging om 4 o 5 Ag/ ml in all cases. 3.4. Inhibi ion o oxin– hy oglobulin an ise um binding by ee oxin F ee glio oxin inhibi s an ise um (an i glio oxin– hy oglobulin) binding o immobilised glio oxin– BSA conjuga e in a ime- and concen a ion-depen- den manne . Fig. 5A shows s anda d cu es ob ained ollowing da a no malisa ion by B/Bo ans o ma ion (Da ies, 1994) p io o plo ing agains ee glio oxin concen a ion be ween 0 and 50 Ag/ml. The s anda d cu es ep esen he esul s ob ained ollowing glio- oxin p e-incuba ion wi h an ise um (glio oxin– hy o- globulin) o 0, 1 and 16 h incuba ion p io o addi ion o mic owells p e-coa ed wi h glio oxin–BSA and he assay hen pe o med as pe Sec ion 2.6. The g ea e deg ee o inhibi ion ob ained ollowing 16 h incuba- ion o ee glio oxin wi h cogna e an ise um sugges s ha ele an IgG exhibi s low a idi y o ee glio- oxin. Mic opla e coa ing a 1 Ag/ml glio oxin–BSA and educ ion in an ise um dilu ion o 1/5000 ( om 1/ 40000) acili a ed a 41% educ ion in an ibody bind- ing a 50 Ag/ml glio oxin (Fig. 5B). Apa om his, al e a ion in assay condi ions h ough he use o al e na i e bu e s/pH has no esul ed in a signi ican imp o emen in assay pe o mance. Nei he has he addi ion o low concen a ions o educing agen s such as di hio h ei ol, which con i ms ha an ibody eac i i y is mos likely no di ec ed agains he educed o m o glio oxin. Fig. 5C shows a s anda d cu e o ee hel olic acid de ec ion by compe i i e enzyme immunoassay as a esul o he inhibi ion o hel olic acid– hy oglobulin an ise um binding o immobilised hel olic acid–BSA conjuga e by ee hel olic acid in a concen a ion-dependen manne . In e es ingly, he ime-dependen inhibi ion o an ise- um binding o immobilised hel olic acid–BSA was no as signi ican as ha obse ed o glio oxin (da a no shown). Thus, in bo h cases, an ibodies aised agains hy oglobulin– oxin conjuga es we e capable o ecognising he ee oxins in compe i i e enzyme immunoassay o ma s. 4. Discussion Con en ional assays o he de ec ion o Aspe gil- lus in ec ion ely upon he iden i ica ion o cell wall componen s eleased by he ungus du ing in ec ion. While hese an ibody-based assays ha e a numbe o applica ions hey can yield alse posi i es and may also ail o de ec in ec ion whe e shedding o cell wall ma e ial has no occu ed (Yeo and Wong, 2002). A numbe o p o ein o non-p o einaceous oxins p oduced by A. umiga us play a c ucial ole in assis ing he ungus o colonise and pene a e pulmo- na y issue and may be de ec ed in blood, u ine o spu um specimens (Ami ani e al., 1995a; Daly and Ka anagh, 2001). As a consequence, we ha e sough o de elop a se ies o an ibody-based immunoassays o de ec ungal oxins p e iously implica ed in issue in asion since hese could ep esen an imp o ed means o iden i ying Aspe gillosis. The wo k p esen ed he e con ains he i s desc ip- ion o he p epa a ion o hy oglobulin-based immu- nogens o he ungal oxins, glio oxin and hel olic acid. We u he desc ibe he gene a ion o sensi i e and speci ic polyclonal an ibodies, which a e capable o de ec ing bo h p o ein-coupled, and ee oxins, and inally he de elopmen o mic opla e-based, compe - i i e immunoassay o ma s o de ec bo h oxins. Thy oglobulin was chosen as he ca ie p o ein o immunisa ion as heo e ically i should con ain a g ea e numbe o amino g oups a ailable o hap en coupling ela i e o BSA (He manson, 1996).Al- hough oxin– hy oglobulin conjuga es could no be eadily cha ac e ised p io o immunisa ion, due o hei la ge size (>660 kDa), he app oach o simul a- neous syn hesis and cha ac e isa ion o oxin–BSA conjuga es by SDS-PAGE and MALDI-TOF mass M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230228 spec ome y con i med ha bo h chemis ies (EDC and PMPI/SATA) employed o p o ein modi ica ion we e success ul. Indeed, he subsequen analysis o all an ise a gene a ed con i med he alidi y o his s a egy. High molecula weigh conjuga e o ma ion was e iden ollowing Wes e n blo analysis o bo h ox- in–BSA conjuga es (Fig. 2B). This is mos likely due o an ibody eac i i y agains oxin modi ied/EDC c oss-linked BSA which was also o med du ing oxin–p o ein coupling. I is possible ha simila high molecula weigh conjuga es we e o med du ing oxin– hy oglobulin syn hesis and may ha e con ib- u ed o he signi ican immunogenici y o esul an conjuga es. Using MALDI-TOF mass spec ome y, Keough e al. (1997) ha e shown he maximum hap en loading o human se um albumin (HSA) o be 17.9 mol ph halic anhyd ide/mol HSA. The alues o hap en coupling o BSA in he p esen wo k a e somewha lowe , and possibly esul om he di e - en ial eac i i y o q-amino g oups in BSA owa ds ac i a ed ungal oxins ela i e o hose in HSA. In e es ingly, addi ion o sulpho-N-hyd oxy succini- mide o he EDC-media ed coupling eac ion did no enhance conjuga e o ma ion (da a no shown). A disc epancy a ose wi h espec o glio oxin loading on BSA, whe eby SDS-PAGE con i med a loading o 15 mol glio oxin/mol BSA while MALDI- TOF mass spec ome y indica ed only 4 mol glio- oxin/mol BSA. P e ious analysis o glio oxin has sugges ed ha he molecule agmen s when mass spec ome y is used as a means o de ec ion (Taylo e al., 1996), hus i is likely ha glio oxin has been clea ed o deg aded om BSA conjuga es esul ing in he appea ance o an inco ec conjuga e m/z a io by mass spec ome y. Thus, we belie e he es ima e o 15 mol glio oxin/mol BSA by SDS-PAGE is a mo e eliable es ima e o hap en loading. Ini ial sc eening o an i oxin– hy oglobulin an i- se a by Wes e n blo ing was supplemen ed by igo - ous mic opla e enzyme immunoassay e alua ion o an ise a i e and speci ici y. Da a p esen ed in Figs. 3 and 4 con i m he speci ici y o wo p epa a ions o an i glio oxin– hy oglobulin an ise a (02-5G and 02- 6G). I is clea ha an ise um 02-5G had a highe i e (Fig. 3) and was un eac i e agains immobilised hel olic acid–BSA conjuga es. I was he e o e used o glio oxin de ec ion by compe i i e immunoassay. Fu he mo e, only glio oxin–BSA, and nei he ee BSA no hy oglobulin, was capable o inhibi ing cogna e an ise um binding o immobilised glio- oxin–BSA (Fig. 4). The IC 50 alues (Fig. 4) ob ained o bo h oxin–p o ein conjuga es (4–5 Ag/ml) equa- es o app oxima ely 70 nM oxin–p o ein and indi- ca es he ela i ely high a ini y each an ise um p epa a ion o p o ein coupled oxin. Wo k by Chan and Ho (2002) has ecen ly dem- ons a ed he u ili y o p o ein-conjuga ed hap ens in elici ing an i hap en–p o ein polyclonal an ibodies. He e, we demons a e ha such an ibodies can be u he u ilised o de ec ee, in addi ion o, conjuga - ed hap ens. A minimum o 15–30 AM glio oxin (5– 10 Ag/ml) was de ec able by compe i i e immunoas- say ollowing o e nigh incuba ion o ee oxin wi h an i glio oxin– hy oglobulin an ise um. This is com- pa able o he lowe limi o sensi i i y (10 AM) ecen ly epo ed by Tuomola e al. (2000) o he de ec ion o 3-me hylindole using an i-3-me hylindole monoclonal an ibodies and high sensi i i y luo ome - ic de ec ion. I can be seen om Fig. 5B ha a 5 educ ion in glio oxin–BSA coa ing le el ( o 1 Ag/ml) acili a ed he de ec ion o ee glio oxin, whe eby a 41% dec ease in an i glio oxin– hy oglobulin binding o immobilised glio oxin–BSA was e iden in he absence o any p e-incuba ion wi h ee oxin. Ini ial a emp s o de ec ee oxins using mic opla es coa ed a sa u a ing concen a ions o hel olic–BSA conju- ga e we e unsuccess ul (da a no shown). Consequen - ly, i was decided o coa mic opla es a lowe coa ing concen a ions o he oxin–BSA conjuga e wi h he in en o enhance he likelihood o an ibody eac i i y wi h ee as opposed o immobilised oxin. This s a egy p o ed success ul. Wi h espec o hel olic acid, no p e-incuba ion wi h espec i e an ise um was equi ed o enable he de ec ion o ee hel olic acid on mic opla es p e-coa ed a 0.05 Ag/ml hel olic– BSA conjuga e (Fig. 5C). Again, a sensi i i y o de ec ion equi alen o 17 AM hel olic acid (10 Ag/ ml) was achie ed. In summa y, hap en conjuga e syn hesis has a- cili a ed he de elopmen o compe i i e immunoas- say o ma s o he de ec ion o he ungal oxins, glio oxin and hel olic acid. Wo k di ec ed owa ds he gene a ion o monoclonal an ibodies agains hese molecules, in addi ion o he assessmen o he esul an diagnos ic po en ial, is cu en ly unde - M. Fox e al. / Jou nal o Mic obiological Me hods 56 (2004) 221–230 229