Food Microbiology, 1987, 4, 147-153

The detection of aflatoxin 5, in peanut kernels, peanut butter and maize using a monoclonal antibody based enzyme immunoassay A. A. G. Candlishl, W. H. Stimson* and J. E. Smith3

1 May and Baker Diagnostics Ltd, Montrose House, George Street, Glasgow Gl 1 YT, UK; 2 Department of Bioscience and Biotechnology, Immunology Division, University of Strathclyde, Glasgow G4 ONR, UK; s Department of Bioscience and Biotechnology, Applied Micro- biology Division, University of Strathclyde, Glasgow Gl lXW, UK.

Received 18 January 1987

A simple procedure was validated for the routine immunochemical analysis of AFBl in peanut kernels, peanut butter and maize. The specificity, affinity and sensitivity of the monoclonal antibody (McAb) employed was such that minimal sample prepara- tion was required. The enzyme immunoassay (EIA) had a sensitivity for standard AFBl of 0.2 ng ml-1 with a working range up to 30 ng ml-1 and was not significantly affected by matrix interference of samples. Essential protocol features were (1) blending substrate with methanol: water; (2) filtering blended sample; and (3) analysis of filtrate by EIA after dilution with buffer. Average recoveries of AFBl spiked samples at levels of 6400 ppb were 90-112&%o. Using laboratory prepared samples contaminated with Aspergillus flavus there was high positive correlation (r = 0.97) when EIA results were compared with thin layer chromatography (TLC) techniques.

Introduction Aflatoxin Bi (AFB,) is a highly toxic and carcinogenic low molecular weight secondary metabolite produced by Aspergillus flavus and A. parasiticus and commonly occurs in agricultural commo- dities such as peanuts, maize, wheat and animal feeding stuffs (Smith and Moss 1985). Determination is normally by thin layer chromatography (AOAC 1984) or high performance liquid chro- matography methods (Hunt et al. 1978). However, such methods are usually time consuming and expensive. The recent development of enzyme linked immuno- sorbent assays (ELISAs) for AI?Bl (Bier-

Please address all correspondence to A. A. Candlish at the above address.

0740-0020/87/020147 + 07 $02.00/O

man and Terplan 1982, Fan and Chu 1984) offer cheaper, more rapid and equally sensitive detection methods. The ELISA methods also allow greater sample throughput without the loss of sensitivity of AFBl detection.

However, these advantages will only be realized if a consistent, standard and reproducible supply of antibody can be obtained. Such sources of antibody known as monoclonal antibodies (McAbs) can now be developed with the advent of hybridoma technology (Kohler and Milstein 1975). To date only poly- clonal antisera specific to AFBl have been described in an ELISA system for the determination of AFBl in peanuts and maize. This present paper reports the application of a direct competitive

0 1987 Academic Press Limited

148 A. A. G. Candlish et al.

ELISA based on a high affinity, specific McAb to the detection of AF’Bl in peanuts, peanut butter and maize. The method requires a single extraction step allowing detection levels of 5 ppb AFB1. The method could be further developed for large scale screening of samples.

Materials and Methods Apparatus EIA reader used was Model MCCl340 (Flow Laboratories).

Materials All inorganic chemicals and organic solvents were at least of reagent grade. AFB1, bovine serum albumin (BSA Fraction V), tetra- methyl benzidine (TMB), hydrogen peroxide (HzOz), horseradish peroxidase (HRP) and Tween 20 were purchased from Sigma Chem- ical Co. (Poole, England). ELISA plates were purchased from Dynatech (‘Micro ELISA’).

Preparation of proteinlAFB1 conjugate AFBi-BSA was synthesized by methods pre- viously described (Candlish et al. 1985). AFBl was converted to the oxime derivative and conjugated to BSA using a water soluble carbodiimide method. A molar ratio of 22 : 1 was obtained as determined by A362 to total protein concentration.

Tissue culture and McAb production This has been described previously (Candlish et al. 1985). The specificity of the McAb was AFGi = 14%, AFBz = 13% and AFGz = 1% by the 50% displacement method. This was determined by using the cross reacting metabolite at various concentrations as the competitor in the ELISA.

Affinity determinations The affinity dissociation constants (KJ of the McAb were determined by an indirect ELISA method of equilibrated McAblantigen solu- tion (Friguet et al. 1985). The McAb from ascites fluid at a dilution of 1: 50 000 was added in equal volumes to various concentra- tions of standard AFBl (0.2 to 30 ng ml-i). This was incubated overnight at room tem- perature, after which 100 pl aliquots of the equilibrated solution was added to AFB1- BSA coated ELISA plates. This was incu- bated at 37°C for 1 h and then washed. To

detect the unequilibrated McAb which was bound to solid phase AFBl a second anti- mouse-y-globulin horseradish peroxidase conjugate was added in 100 ~1 volumes to each well and incubated 1 h at 37°C. This was washed and enzyme activity determined. The fraction of McAb bound to AFBl was calcu- lated by AdA,-A where A, = AAs0 with zero equilibrated AFBl and A = Ads0 with various AFBl concentrations equilibrated with McAb. This was plotted against the concen- tration ofAFBl in moles at each A value. The concentration of McAb was consistent throughout.

Preparation of McAb : HRP conjugates McAb : HRP conjugates were achieved using the periodate method for protein : protein conjugation. HRP (5 mg) in 0.45 ml 0.1 M

NaHC03 was activated with 0.05 ml 0.05 M

sodium m-periodate solution (in distilled water) and incubated for 2-3 h in complete darkness. The activated enzymes were added to a closed Pasteur pipette simultaneously with 0.33 g Sephadex G-25 and 1.5 ml anti-AFBi McAb (10 mg ml-i). This was incubated for a further 2-3 h before elution of conjugate with 0.1 M carbonate buffer, pH 9.3. The conjugate was then stored in small aliquots at -20” (Kurstak, 1985).

Spiking and extraction of samples for ELISA For spiking studies, AFBl was dissolved in methanol (100 pg ml-l) and added to log of ground sample to give final concentrations of 6, 20, 30, 60, 90 and 120 ppb for maize; 40, 100,200,400 ppb for peanut kernels, and 20, 30, 60 and 90 ppb for peanut butter. The methanol was allowed to evaporate before extraction. Naturally contaminated samples were produced in the laboratory by growing Aspergillus flavus strain NRRL 5774 on maize or peanuts and mixing the heavily contaminated samples with uncontaminated samples. This gave a source of contaminated samples similar to those found under natural conditions, i.e. fungal produced mycotoxins and not artificially spiked samples.

Each 10 g of naturally contaminated or spiked sample was shaken in a stoppered flask with 50 ml methanol : water (55 : 45) and 20 ml hexane containing 0.4 g NaCl. The blended substrate was filtered through a Whatman No. 4 filter paper and the filtrate diluted with 0.1 M Tris/HCl, pH 8.5; 1: 4 for maize and 1: 8 for peanut kernels and peanut

Enzyme immunoassay for Aflatoxin 6, 149

butter. The diluted filtrates were then applied directly to the competitive ELISA.

ELISA protocol The ELISA procedure employed was the direct competitive method carried out in microtitre plates. In practice, AFBi-BSA coated microtitre plates (05 ug ml-i ofAFBi coated overnight in 0.02 M TrisHCl, pH 9.2 at 37”) were washed five times with 0.2 M Tris/HCl, pH 7.4 containing 0.2 M NaCl and 0.05% Tween (wash buffer). Standard samples of A.FBl (0.05 ml) were added to appropriate plate wells prior to the addition of McAb-HRP conjugate (O-1 ml). Plates were incubated at 37” for 1 h, emptied and washed five times as before. Enzyme substrate (TMB) 0.15 ml) was added and incubated 0.5 h in darkness at room temperature. The reaction was stopped by the addition of 2 M HeSO (O-05 ml) and Au0 measured. This was plot- ted against the logic of AFBl concentration (ng ml-l) and quantitation of AFBl in samples was by reference to the standard curves.

Effect of methanol on competitive ELISA The effect of methanol on the standard dose response curve for AFB1, present in 0, lo,20 and 50% methanol, was determined by ELISA.

Results Affhity &terminations The Kd value of the McAb cell line (4El) used throughout these studies was found to be 2.01 x 10-s ~-1 as determined by the Klotz plot in Fig. 1.

Validation of AFBl ELISA for application to sample analysis The intial criteria for the validation of a direct competitive ELISA for AFBl was the construction of a suitably sensitive standard curve. From Fig. 2 it can be seen that McAb based ELISA described had a sensitivity of O-2 ng ml-l and a working range up to 30 ng ml-l for A.FB1. Peanut kernels, peanut butter and maize extracts prepared as described from known AFBl negative samples did not show significant interference in the

201 I

5 I5

I.Or T I I I 0 I 2 3

l/M x 108

Fig. 1. Klotz plot of the binding of AFBl to McAb produced from hybridoma cell line 4El by the ELISA method with impure Abs from tissue culture fluid. V is the fraction of bound Ab.

I IO 50

ng ml-’ compet Ing AFB,

Fig. 2. Dose response curves for AFBl by direct competitive ELISA with standards prepared in diluted water : methanol extract of peanut kernels and bufffer (0.1 M Tris/HCl, pH 8.5). (0 Buffer; x 1:8 negative sample extract. n 1: 4 negative sample extract.)

ELISA. Table 1 illustrates the interfer- ence caused by sequential dilution of peanut butter extracts in the ELISA. Figure 2 shows the lack of matrix effect on the standard dose response curve for AFB1, i.e. the standard curve for toxin in assay buffer was identical to that obtained by diluting standards in sample extract. However, if lower dilutions of sample extracts were taken, the dose response curves were not super- imposable, i.e. at 1:4 dilution for peanuts and peanut butter.

150 A. A. G. Candlish et al.

Table 1. Interference of water : methanol peanut butter extracts prepared by the BF method to determine AR’& by direct competitive ELISA.

Dilution of extract

in buffer A450

Percentage Interferences

1:2 1,448 -28.5 1:4 1.890 -5.3 1:8 1.934 -3.1 1:16 1.996 0 1:32 1.957 -2 1:64 1.998 0

Buffer 1.995 0

a Defined as percentage ratio of AabO of diluted extract in buffer to AdeO of buffer in ELISA. Buffer 0.1 M Tris/HCl, pH 8.5. N.B. Similar results were obtained with peanut kernel extracts. With maize extracts the interfer- ence was reduced to an insignificant level at a dilution of 1: 4 with buffer.

ELISA of spiked samples The ELISA results obtained on samples spiked with AFB1 are shown in Table 2. The average recovery for peanut kernels was 97-108%, for peanut butter 90- 100% and for maize 98-1126%. The standard deviations throughout all experiments were consistently low.

Correlation of ELISA with TLC Samples of maize, peanut kernels and peanut butter naturally contaminated with AFBl were analysed by ELBA and TLC and a comparison made of the analytical data (Table 3). AFBl content of samples was determined by ELISA by directly comparing competition caused by methanol: water diluted extracts of samples to a standard competition curve for AFBl in buffer. A highly significant correlation coefficient of r = 0.97 was obtained when the analytical results of ELISA were compared with TLC. Table 4 illustrates typical coefficient of variabili- ties obtained by ELISA analysis ofAFB1 present in peanut kernels and maize. These varied from 4.7% for AFBl detec- tion at 75 ppb to 21.5% for detection at 7.1 ppb.

Effect of methanol on competitive ELISA Figure 3 illustrates the effect of methanol on the standard dose response curve for AFBl as determined by direct competitive ELISA at various concentra- tions.

Table 2. Recovery of AFT& from spiked samples of peanut kernels, peanut butter and maize as determined by direct competitive ELISA using a water: methanol extraction procedure (BF method) diluted in buffer and then directly applied to the ELISA.

Substrate ppb added

(spiked level) ppb detected

by ELBA 1 S.D.

(n = 6) Percentage

recovery

Peanut kernels

Peanut butter

Maize

40 43.2 k14.6 108 100 104 k18.7 104 200 194 -+ 14.0 97 400 432 k20.3 108

20 20 + 4.4 100 30 27 -+ 4.1 90 60 55 + 3.5 91.7 90 85 + 3.2 94.4

6 6.2 If: 1.4 103 20 20.4 + 1.9 102 30 29.7 + 1.8 99.2 60 59 + 4.2 98 90 90 + 7.2 100

120 135 + 8.1 112.5

Enzyme immunoassay for Aflatoxin B, 151

Discussion This laboratory has previously described the development of a McAb to AFBl and its application to a direct competitive ELISA to determine AFBl in standard solution (Candlish et al. 1985). No results were given on the validation of this assay for the detection of toxin in natural samples. Other groups have vali- dated indirect competitive ELISA methods for AFBl (Fan and Chu 1984, Morgan et al. 1986) where anti-AFBi polyclonal antibody competed with free toxin for binding to a solid phase AFB1- protein conjugate. A second antispecies enzyme conjugate was then required to determine total bound antibody.

In the direct competitive ELISA des- cribed here, McAb specific to MB1 was conjugated to HRP enzyme and added to the AFB,-protein coated wells after the addition of AFBl standards or samples free in solution. The McAb-HRP conju- gate was incubated and competed for solid phase AFBl and free AFB1. Another direct competitive approach is to coat microtitre plates with antiserum specific to A.FBl and add simultaneously standard or sample MB1 along with AFBl bound to an enzyme. The solid phase antiserum then binds competi-

tively with free toxin either bound or unbound to an enzyme (El-Nakib et al. 1981). The direct assay approach is preferable because it requires one less incubation step as well as one less wash

Table 3. Correlation of direct competitive ELISA with TLC (BF method) for quanti- tation of AFB, in laboratory contami- nated samples of peanut kernels, peanut butter and maize.

Substrate

TLC ELISA analysis analysis

hvb) (ppb)

Table 4. Variability of peanut kernels and maize water:methanol extract samples analysed by direct competitive ELJSA.

Maize 25 26.7 14 16.2 10 12.2 14 13.5

Peanut kernels 100 110 70 75

5 7.1 10 17 40 30

135 300 350 480

Peanut butter 50 55 12 16 10 10 0 0

r = O-97, i.e. high positive correlation. TLC results were the average of duplicate analysis.

Peanut kernels Maize Replicate sample

analysis Sample 1 Sample 2 Sample 3 Sample 4

1 72.5 9.5 16 26 2 75 6-5 13 3 70 6.5 15 28.5 4 77.5 5.0 10.5 30 5 81 6.0 13 25 6 76 - 24

Mean* 75 7.1 13.5 26.7

R% 3-5 4.7 21.5 1.5 14 1.9 2.2 8.3

a Concentrations of AFBl are all in ppb.

152 A. A. G. Candlish et al.

ng ml-‘competmg AFB,

Fig. 3. Effect of methanol on the dose response curve for AFBl by direct competi- tive ELISA. The values for 10% and 20% methanol are superimposible with buffer results. (m 50% methanol; 0 Buffer.)

step than the indirect assay and there seems to be little or no loss in sensitivity. Of the two direct assay procedures avail- able, the one described in this paper is undoubtedly superior as no toxin- enzyme conjugates are required. Such conjugates are often unstable and require the synthesis of precise molar ratios to give adequate assay sensitivi- ties which may lead to inefficient conju- gation (Fan and Chu 19843. In contrast, the conjugation of McAbs and polyclonal antisera to enzymes has been well evalu- ated and described (Kurstak 1985).

The McAb used in this ELISA has a low & value for AFBi indicating that it is highly sensitive and specific to AFB1. This has been verified by competition data previously described (Candlish et al. 1985). The level of detection by ELISA was compatible with TLC and although HPLC methods can detect down to O-2 ppb, they are generally not suitable for screening purposes. The present assay will be particularly useful

for distinguishing positive AFB contain- ing samples from negative samples as well as producing a semi-quantitative result.

Although many other types of ELISA have been described for AFBl few have found widespread application and the potential of immunological methods has not yet been fully realized. This has probably been due to the small and limited supply of adequately active antisera. However, this problem has now been overcome by the development of a McAb to AFBl described in this paper with a similar sensitivity to that of polyclonal antisera for the toxin as deter- mined by ELISA (Biermann and Terplan 1982, Fan and Chu 1984, Morgan et al. 1986).

The AFBl extraction procedure employed gave no problems in relation to non-specific matrix effects on the ELISA (Fig. 2). Furthermore, methanol at con- centrations of 20% had no significant effect on the ELISA and even at 50% methanol a dose response for AFBl could be constructed although AJhO values were elevated. The simple extraction procedure allowed a high throughput of sample determination by ELISA.

The recovery and reproducibility of the ELISA for AF& analysis can be seen in Table 1. A high recovery with reprodu- cible results was obtained due to the sample extraction and analytical tech- nique.

The present results are very promising but full evaluation of the ELISA, in at least a commercial aspect, will require the determination ofAFBl in samples by ELISA and TLC or HPLC both within the laboratory of invention and through- out many different laboratories.

Enzyme immunoassay for Aflatoxin /3, 153

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