Analyyhca Chumca Acta, 275 (1993) 341-345 Elsewer Science Pubhshers B V , Amsterdam

341

Development of an immunoaffinity column and an indirect immunoassay with a biotin-streptavidin

detection system for aflatoxin M, in milk

C De Boevere and C Van Peteghem

Laboratory of Food Analym, Uruverstty of Ghent, Harelbekesmat 72, %WO Ghent (Belgud

(Received 20th May 1992, rewed manuscnpt recewed 21st October 1992)

A sensmve and rapId screemng method for the estnnation of atlatoxm M, (APM,) 111 mdk has been developed Mdk samples were first purd’ied by mnnunoaffin~ty chromatography (IAC) usmg polyclonal ant&o&es ram~I m rabbits agamst ailatoxm MI-bovme serum albumm &FM,-BSA) and coupled to an a-ted sepharose matnx. The eluate was analyzed III an mdnect competitwe streptawdm-blotm modtied enzyme bnked mummoassay (EIA) Mnzrotltre plates were coated wrth APM,-BSA that competes mth the analyte m the sample for bmdmg with the blotmylated a&body Bound blohnylated antlbody was detected usmg a streptavldm blotmylated horseradish peromdase complex The llrmt of dete&on of the EXA IS 2 ng kg-’ By combmatlon of IAC and EIA, spiked nulk samples were analyzed In conclusion, IAC-EIA 1s a sensltwe and rapld method for the estimation of APM, m nulk.

Key~r& Immunoassay, Atlatoxms, Blotm, Mdk, Streptavrdm

Aflatoxm B,, Bz, G, and G, are frequently occurrmg contammants m human food and am- mal feed [l] AFM, 1s a hydroxylated metabohte of aflatoxm B, (AFB,) whch IS found m the mdk of ammals fed AFB, contammated feed The carryover of AFB,, mgested by feed, mto AIM, m the mdk was expernnentally determmed and 1s l-2% It varies from ammal to ammal, from day to day and from one mdkmg to the next 121 Because of the tomcIty of aflatoxms and the nn- portance of rmlk m the human dret, especmlly for mfants and young chddren, mdk has the greatest demonstrated potential for mtroducmg aflatoxm residues from edible animal products mto that &et [3] Many countries have regulated the

Correspopldme to C De Boevere, Laboratory of Bromatol- ogy, Umvers@ of Ghent, Harelbekestraat 72, 9000 Ghent (Belgium)

amount of AFM, m dauy products A tolerance level of 0 05 fig kg-’ IS generally accepted

It was our arm to develop a rapid prepunfica- tlon system and a sensitive assay with mnumum mterferences from the matrw

EXPERIMENTAL

The nucrotitre plate photometer was from Eu- rogenetlcs (Tessenderlo) The rmcrotitre plate washer was from Flow (Helsti) Radloactmty was counted m a Rackbeta Model 1711 hquld scmtdlatlon counter LKB Wallac (Turku)

Materials All reagents were analytxal grade AFM,-

oxune-BSA, aflatoxm M, @FM,), aflatoxm Bz

342 C De Boevere and C Van Peteghem /Anal Chun Acta 275 W93) 341-345

(AFB,), aflatoxm G, (AFG,), aflatoxm G, (AFG,), aflatoxm Q1 (AFQ1), aflatoxlcol (AFOL), bovme serum albumm (BSA), tetramethylbenzl- dme (TMB), blotme-e-ammocaproyl-N-hydroxy- succmnmde were purchased from Sigma (Mont Lotus, Brea, CA) AFB, and AFM, standards m chloroform (1 kg ml-‘) were kmdly supplied by Dr H P van Egmond, RIVM (Bdthoven) PD-10 columns, CNBr-activated sepharose, Dextran T-70 were purchased from Pharmacla (Uppsala) Rabbits were from Laboratolre d’Hormonologle (Marlowe) 3H-Aflatoxm B, (3H-AFB,) with a spe- cific actrvrty of 18 Cl mmol-’ was from Moravek (Brea, CA) Streptavldm blotmylated horseradish peroxldase complex was from Amersham (Ghent) Filter columns (1 ml> were purchased from Baker (Deventer) Mlcrotltre plates Max~sorp were from Nunc (Roskdde) Scmtrllatlon fluid RIALUMA was from Lumac (Olen) Pro&n 300 was a g& from Rhomn and Haas (Antwerp) Dunethylsul- foxlde, dnnethylformanude, NaCl, Na,HPO,, H,O,, W-L,),SQ, and citric acid were from Merck (Darmstadt)

Productwn and charactenzatwn of antdndes Specific polyclonal antibodies were raised m

rabbits Four rabbits (New Zealand) were unmu- wed by multiple site mtradermal lnJectrons of 250 Fg AFM,-oxune-BSA dissolved m 0 5 ml of saline (0 154 M NaCl), emulstied vvlth 0 5 ml Complete Freund’s adjuvant Two booster mJec- tlons were admmlstered vvlth an interval of 2 weeks Afterwards, 4 mjectlons were admmls- tered at 4-week intervals For the booster mjec- tlons the same emulsion was administered as for the first unmumzatlon Ten days after mjectlon blood was taken from the marginal ear vem Antibodies were obtained as described by Harder and Chu [4] Antibodies were precipitated thrice with (NH,),SO, at a final saturation of 33 3% The precipitate was redissolved m 001 M PBS (0 01 M Na,HPO,, 0 15 M NaCl, 0 02% Pro&n 300, pH 7 4) to the volume of the ongmal serum sample and applied to a PD-10 c&unn as de- scribed by the manufaturer The antibodies were stored at -20°C

Antibody tltres of the different antisera frac- tions of the rabbits were determined by radloun-

IO00 log antiserum titre

IL t1 t I t I t I r

0 20 40 80 80 100 120 140

lmmunlsatlon time (days)

Fu 1 Antibody t&es of 4 rabbits unmund w~tb AFM, Key to symbols n =V6, + =V7, * ==VS, q -V9

munoassay (RIA) The tltre was defined as the reciprocal of the antibody dllutlon required for 50% bmdmg of the tracer A protocol described by Evrard et al [5] was followed Appropnate dilutions of the drfferent purlfled antisera frac- tions were made m RIA buffer (0 2 M Na,HPO,, pH 7 4, containing gelatm, 5 g 1-l) 50 ~1 of the antibody dllutlon and 50 ~1 of 3H-AFBl (10000 cpm or 0 92 run01 3H-AFB,) were added to 150 ~1 of RIA buffer The tubes were incubated at 37°C durmg 30 mm and subsequently for 2 h at 4°C 250 ~1 of dextran coated charcoal (Nont Charcoal, 5 g l-‘, dextran, 05 g 1-l m RIA buffer) was added and the tubes were mcubated for 15 mm at 4°C After centrfigation at 4000 t-pm for 10 mm the supematant was added to 2 5 ml of scmtlllatlon fluid

In Fig 1 the tltres of the dtierent antibody fractions for each rabbit are plotted agamst the lmmumzatlon schedule The booster mjectlons are indicated by arrows

Preparation of an UnmunoajJGuty column Antibodies were coupled to a CNBr-activated

Sepharose matrix according to the mstructlons of

C De Boevere and C Van Pete&em /Anal Chtm Acta 275 (1993) 341-345 343

the manufacturer Finally the gel was equlh- brated wth 30 ml PBS Immunoaffimty columns were made contammg 0 5 ml (capaaty of the gel was 2 5 pg AFM, ml-’ gel) 111 filter cohunns and stored at -4°C

Bwtmylatwn of antrbodm Antibodies were blotmylated as described by

Strasburger and Koben [61 The purlfled un- munoglobulmes were dialyzed against PBS overnight at 4°C The IgG were diluted to a concentration of 1 mg ml-’ with PBS A 2-ml portion was made slightly basic by addmg 90 ~1 of 1 M sodmm phosphate, pH 9, and 20 ~1 of blotm-e-caproyl-N-hydroxysuccmunlde dissolved m dlmethylformamlde at a concentration of 1 mg per 40 ~1 was added with stu-rmg The murture was stirred for 6 h at room temperature and dialyzed against PBS The blotmylated antibodies (AB-B) were stored m fractions of 1 ml at - 20°C

Streptavuim-btotzn based ELISA An indirect competitive EIA was developed

Mcrotltreplates were coated with AFM,-BSA 50 ~1 of AFM,-BSA m PBS (1 pg ml-‘) was added to the wells and the plates were dned overnight at 37°C and stored at -20°C until use The plates were washed 4 tunes wth washing solution (Tween 20, 0 5 g 1-l m PBS) To reduce the non-specific bmdmg 200~~1 ahquots of 3% BSA dissolved m PBS was added to the wells and the plates were left for 30 mm on a shaker The solution was removed and the plates were washed 4 times Standard solutions were made 111 PBS- methanol (9 + 1) 50 ~1 of the standards and 50 ~1 of a 2 4 x 10F6 dllutlon of AB-B m assay buffer (0 1% BSA 111 PBS) were added to the wells m duplo The plates were mcubated overnight at 4°C The plates were washed 4 times and 100 ~1 streptavldm blotmylated horseradish peroxldase complex diluted 10e3 m assay buffer was added and reacted for 30 mm After washing the plates 6 tunes with washing solution, the plates were rinsed with water before adding 100 ~1 of substrate solution (substrate A 0 1 M Na,HPO,, 1% H,O, adjusted to pH 55 with citric acid, and substrate B 0 5 g tetramethylben- zldme dissolved m 40 ml of dunethylsulfoMde,

diluted with bldlstllled water to 1 1 and adjusted with citric acid to pH 2 4, before use equal vol- umes of A and B were mixed) The plates were left for 15 mm on a shaker at room temperature 50 ~1 of 1 M H,SO, was added and the colour was read wth a mlcrotltreplate photometer at 450 nm

Sample preparation A lo-ml volume of skunked milk was diluted

to 20 ml with PBS and applied onto the IAC The column was washed with 18 ml of PBS and the toxms were eluted with 5 ml of PBS-methanol (1 + 9, v/v) The eluate was evaporated to dry- ness under reduced pressure and the residue was dissolved m 5 ml of PBS-methanol (9 + 1, v/v) The IAC was equlhbrated with 20 ml of PBS

RESULTS

In Fig 2 a standard curve for AFM, obtamed by the competitive EIA mth a workmg range between 0 25 pg per well (90% B/B,) and 125 pg per well (20% B/B,) 1s shown The detection limit, defined as the mmunum quantity of the analyte which yields a signal two standard devla- tlons below the signal for zero pg on the standard curve was 2 ng kg-’

The cross-reactivity of dtierent aflatoxm ana- logues was determmed by the EIA Standard solutions of the different cross-reacting toxms WW,, AFBl, AFB,, AFG,, AFGz, AFQ, and AFOL) were made and were added to the wells

%B/e, 90 80 70 60 50 40 30 al D

a2505 1 25 5 la 25 50 xx) 250 500 loxl

Am, pg well'

FUJ 2 Aftatoxm M, standard curve

344 C De Boevere and C Van Peteghem /Anal Chm Actu 275 (1993) 341-345

The protocol described above was followed The results were then plotted m a series of standard curves The cross-reactnnty was arbltranly de- fined as the ratio of the weight of the specfilc antigen (AFM,) required to reduce bmdmg by 50% to the weight of the cross-reactant required to reduce bmdmg by 50%, multlphed by 100 171 In Fig 3 the cross-reacting curves are shown The antibody was specific for AFM,, the cross-reactlv- ltles were 10 26%, 29 62%, 0 34%, 5 70%, 0 21%, 0 60% and 0 10% for aflatoxm M,, B,, B,, G,, G,, Q1 and aflatoxxol, respectively Recovery expenments were done by sp&mg sknnmed milk (fat content I 3 g kg-‘) at different levels of contammatlon 0 05 pg kg-l, 008 pg kg-‘, 0 1

l.lg kg-‘, 0 12 pg kg-l and 0 15 pg kg-’ The results are presented m Table 1

In conclusion, a high sensitive and rapid

L WE 11 -

l-

05-

TABLE 1

Recovery expenments of atlatoxm M, contammated mdk

AFM, spiked AFM, by ELBA Coefficient Number of into milk kg kg-‘) of vanation samples (ag kg-‘) (o/o)

005 0 049 17 8 9 008 0082 66 4 0 10 0 1049 126 8 0 12 0 117 29 3 0 15 0 136 13 4 5

screemng method for AFM, was developed By the use of the IAC up to 30 samples could be purified m one day and analyzed by EIA Only a lmuted number of spiked milk samples have been tested, the method ~11 be further vahdated by analyzmg naturally contammated milk samples

Fig 3 cross-reactmty curves of the antlserum raised agamst AFh+BSA wth other afkwon anakwes GY to symbol a, MG2,

b, AFQI, c, AFM,, d, AFG1, e, AFJ& f, AFW, g, AlW, h, AFQL

C De Boevere and C Van Peteghem /Ad Chm Acta 275 (1993) 341-345 345

This work was supported by the Instttuut voor Wetenschappelgk Onderzoek 111 NlJverheld en Landbouw from which the first author received a scholarship

REFERENCES

1 HP van Egmond, m H P Van Egmond (Ed 1, Mycotoxms III Dauy Products, Elsewer, Amsterdam, 1989, p 16

2 D S P Patterson, EM Glancy and B.A Roberts, Food Cosmet Toxwol , 18 (1980) 35

3 R Jackman, J SCI Food Agrx ,36 (1985) 685 4 W 0 Harder and F S Chu, Expenenta, 35 (1979) 1104 5 P Evrard, P Gaspar and G Ma&m-Romter, J Im-

munoassay, 7 (1986) 313 6 J Strasburger and F Kohen, Methods Enzymol , 184 (1990)

481 7 B A. Morns, m BA. Morns and MN Chfford (Eds 1,

Immunoassay m Food Analysts, Elsevler, Amsterdam, 1985,

P 21