Immunoassays for Residue Analysis - ACS Publications - American

Chapter 13

Downloaded by STONY BROOK UNIV SUNY on December 14, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch013

Detection of Dieldrin by Enzyme-Linked Immunosorbent Assay in Some Dairy Products Mohamed A. Morsy, Azza A. Ibrahim, and Mahmoud M . Hewedi Department of Dairy Technology, Faculty of Agriculture at Fayoum, Cairo University, Egypt

An enzyme-linked immunosorbent assay (ELISA) was used to monitor the distribution of dieldrin in Domiati cheese, Cheddar cheese and Yogurt. The effect of the salt concentration (0-10% w/v) on the distribution of aldrin/dieldrin between curd and whey in Domiati cheese was studied. There was a positive correlation between the amount of salt added to the milk and the amount of aldrin/dieldrin detected in the whey. On the other hand, in cheddar and yogurt filtrate (acid whey), aldrin/dieldrin were generally not detected.

The use of pesticides has contributed in a large part to the high production levels which modern agriculture in developed countries have been able to achieve. Egypt used 61,705 metric tons of pesticides in the period between 1952 and 1984 (7), nearly 50% of this quantity found its way into the soil and is a permanent source of persistent pesticides (2,5). Measurable amounts of pesticide residues in our food including dairy products present a variety of problems. For example organochlorine insecticides which have been in use for more than four decades in agriculture and public health programs all over the world, have caused one of the most serious environmental problems and are commonly detected in air, soil, aquatic and terrestrial wild life, and in different food items (4-7). The organochlorine insecticides are characterized by their capability to travel great distances from application sites, and they are sufficiently stable and retain their identity for years, they tend to concentrate in fatty tissues of higher members of the food chain. Several organochlorine residues including DDT and dieldrin, were detected in the liver and adipose tissues of humans at higher levels than other pesticide groups. Food contaminated with pesticide residues are reported to be the major source of human's daily intake. Milk, milk based formula, and its by-products contain a profound amount of pesticide residues (6). Animal feed, feed mixtures and fodder grasses have been shown to be the major source of contamination of animal tissues and milk and milk products (8-11). Studies have clearly demonstrated the transfer of pesticide residues from animal food 0097-6156/96/u621-O176$15.00A) © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Detection of Dieldrin by ELISA in Dairy Products

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stuffs into the tissues of farm animals (72). Further, organochlorine insecticides have been the pesticides found most frequently in milk and dairy products (13-20). Chromatographic methods have commonly been used to analyze pesticide residues in food stuffs (17,18,21-23). These methods are time consuming and laborious, tedious, require personal skills and are expensive. The existence of immunoassays for analysis of pesticide residues such as aldrin/dieldrin provides a relatively cheap, sensitive, reliable and rapid assay which can be used to monitor food samples routinely. The present paper was developed to examine the distribution of pesticides, parathion, diflubenzuron and dieldrin spiked into milk and processed into different dairy products. Materials and Methods Control Samples of Dairy Products. Control samples were made from cow's milk as follows: Domiati cheese. Pasteurized milk, 37 °C Ψ Salting (0, 2.5, 5, 7.5, 10%, w/v) Ψ Coagulation (Renneting), 37 °C Ψ Draining of whey (whey was collected) Ψ Packaging

Cheddar cheese. Pasteurized milk, 30 °C Ψ Addition of starter 1.5% (30 rnin/30 °C) Ψ Coagulation (Renneting), 30 °C Ψ Cutting of curd (whey acidity 0.15%) Ψ Gently stirring (10 min) and scalding of curd (up to 40 °C in 60 min and whey acidity 0.18%) Ψ Draining of whey (at acidity 0.19%) and whey was collected Ψ Cheddaring (curd acidity 0.85% in 3 h) Φ Milling Ψ Packaging and pressing

Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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IMMUNOASSAYS FOR RESIDUE ANALYSIS


Yogurt. Milk Ψ Heat treatment (90 °C/15 min) Ψ Cooling to 40 °C Ψ Inoculation of the starter (3%) Ψ Incubation (40 °C/3 h) Ψ Cooling of yogurt (4 °C) Ψ Filtration of yogurt, Yogurt filtrate (acid whey)

Spiking of the M i l k Samples. Dieldrin was dissolved in methanol and added to the milk to reach a concentration of 5 μg/mL. Preparation of Cheese Extract. Freshly prepared cheese curd was mixed with distilled water (1:2) in a blender for 5 min at room temperature. The homogenate was centrifuged at 3000 rpm and the supernatant was used as the cheese extract. Preparation of Whey from Y o g u r t Yogurt curd was carefully stirred using a spatula and filtered using filter paper (Whatman No. 1), and the filtrate was collected. Preparation of Immunogen (Figure 1) Functionalized Hapten. 6,7-Dihydro-6-carboxyaldrin was prepared as described by Langone and Van Vunakis (24), by heating a mixture of hexachlorocyclopentadiene (10 g) (Aldrich Chemical Co., Milwaukee, WI) and bicyclo-(2.2. l)-5-heptene-2carboxylic acid (5.0 g; 36 mmol) (Lancaster Synthesis Inc., Windham, NH) at 90 °C for 48 h. The black semi-solid was dissolved in hot ethanol (150 mL) and an insoluble solid was separated by filtration. The filtrate was concentrated to dryness in vacuo and the residue was triturated with petroleum ether to give approximately 4.0 g of tan solid. Two recrystallizations from chloroform-petroleum ether gave a white powder. The melting point of the product was 216 °C. Preparation of Conjugates for Immunization. To 100 mg (0.24 mmol of the hapten derivative and 70 mg (0.61 mmol) N-hydroxysuccinimide dissolved in 1.0 mL dimethylformamide (DMF) was added 60 mg (0.29 mmol) of Jv^Vdicyclohexycarbamide (DCC). After 30 min, precipitated JV^-dicyclohexylurea was separated by centrifugation. A 0.01 mL aliquot of the supernatant liquid in 1.0 mL dioxane was added to 60 mg of bovine serum albumin (BSA) dissolved in 3.0 mL, water p H 7.2. After standing at 4 °C overnight, the reaction mixture was dialyzed




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exhaustively against 50% dioxane-water, then against 0.005 M phosphate, 0.15 M sodium chloride, pH 7.2.


Antibody Production and E L I S A Assay. New Zealand white rabbits (20 weeks old) were immunized with a solution of conjugate (prepared in water) mixed with an equal volume of Freund's complete adjuvant and maintained by booster injections of antigen in Freund's incomplete adjuvant at three months intervals. Each rabbit received a total of 2 mg per 1.0 mL of immunogen injected subcutaneously into the neck flap at five sites (200 pL per site). Blood samples were collected from the lateral ear vein at monthly intervals and tested for antibody production using ELISA. Purification of Antibody. Positive serum samples were treated by affinity chromatography to remove bovine serum albumin antibodies. CNBr-sepharose 4B (Pharmacia, Piscataway, NJ) was linked to B S A as per manufacturers handbook. Serum samples were diluted 1:1 in loading buffer (0.1 M phosphate, 0.5 M NaCl, pH 7.2) and aliquots (5 mL) loaded onto a 10 cm column filled with swollen gel. The unbound fraction containing anti-pesticide antibodies was eluted with loading buffer. The B S A reactive fraction was subsequently recovered by eluting the column with Glycine-HCl (0.1 M ) pH 4.0. Microtitre Plate Sensitization. Dynatech Immunol Π 96-well microtitre plates (Dynatech Laboratories Ltd., Billingshurst, Sussex, U K ) were coated with the conjugate (5 pg/mL in 0.1 M carbonate/bicarbonate buffer, pH 9.6, 0.1 mL per well). After overnight incubation at 4 °C the plates were washed three times with coating buffer, emptied and dried at 37 °C. Finally the plates were sealed with tape and stored at 4 °C. Non-Competitive Indirect E L I S A for the Detection of Antibody. Serum samples were tested for antibody by means of a non-competitive ELISA. Serial dilutions of the antibody, in the range of 1:10 to 1:10 were prepared in wash buffer containing 1% bovine serum albumin (0.01 M phosphate containing 0.5 M NaCl, 0.5% Tween 80, 0.01% Thimerosal, pH 7.8). Samples of each dilution (0.1 mL) were added in duplicate to wells of a sensitized microtitre plate. The negative control was dilution buffer. The plate was then incubated for 1 h at room temperature with constant shaking, emptied, washed 5 times with wash buffer and blot dried. Goat anti-rabbit IgG peroxidase conjugate (0.1 mL) (Sigma Chemical Co., St. Louis, MO), diluted 1:3000 in wash buffer was added to each well. The plate was then incubated 1 h, washed 5 times and blot dried as before. The substrate was prepared from a stock solution of 2,2'-azino-bis(3-ethylbenzthialozine-6-sulphonic acid (ABTS) 20 mg/mL in water. Substrate solution (0.15 mL ABTS stock solution + 4.85 mL citrate buffer 0.1 M citrate monohydrate, pH 4.0 with 0.05 mL of H 0 (30%)) 0.1 mL was added to each well. Plates were incubated, with shaking at room temperature. The absorbance was measured at 405 nm on a Titertek Multiscan (Flow Laboratories Ltd., Rickmansworth, Hertfordshire, UK). Samples recovered following removal of B S A reactive fractions were tested in the same way in order to determine their optimum working dilution prior to use in the competitive ELISA. 5

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Indirect Competitive E L I S A Procedure. Analysis of prepared pesticide samples was conducted by competitive ELISA. Dieldrin (Fluorochem Ltd., Derbyshire, U K ) was dissolved in methanol (1 mg/mL) then different concentrations were prepared by dilution in control whey or cheese extracts in Domiati cheese (0, 2.5, 5, 7.5 and 10% salt concentration), cheddar cheese and acid whey containing 1% BSA, to reduce non-specific reactions. Dilutions were prepared such that the final concentration of methanol was always less than 1%. Prior analysis indicated that methanol up to 10% did not interfere with binding of the antibody to the coated wells. Pesticide samples (50 pL) were added to wells of the sensitized plates. Purified antibody (50 pL) was added to each well containing pesticide solution and to each well containing buffer or milk (50 pL) without pesticide which were included to provide a positive control (maximum antibody binding). Plates were incubated for 1 h at room temperature with constant shaking. The subsequent steps were the same as for the antibody detection ELISA described previously. Measurement of the absorbance at 405 nm of the test and control samples indicated the degree of inhibition of antibody binding produced by the test samples. Results and Discussion The molecular weight of the aldrin/dieldrin hapten is too low to initiate an immune response (25). It was therefore necessary to conjugate the dieldrin hapten to a larger carrier protein. Rabbits were immunized with the conjugate and the antiserum used to develop an ELISA capable of detecting pmol levels of dieldrin. The serum contained antibodies which reacted with either the carrier protein (BSA) or the hapten, therefore affinity chromatography was used to remove the B S A reactive antibodies. The pesticide used in this study is poorly soluble in water, therefore, methanol was used as the primary solvent. To ensure that the presence of methanol in the assay did not interfere with antibody binding, buffer plus methanol over a range of 0-10% methanol was tested. No effect was detected over this range of methanol concentration. Standard pesticide dilutions were prepared by dissolving the analyte in methanol and diluting the resulting solution with whey containing salt in the range of 0, 2.5, 5, 7.5, 10% (Figure 2), Domiati cheese (0, 2.5, 5, 7.5, 10% salt) extracts (Figure 3), Cheddar cheese extract (Figure 4) and Cheddar cheese whey (Figure 5). Domiati cheese is commonly used in Egypt. This type of cheese differs from the other types in salting method. The salt is added to the milk (pasteurized or raw) at high levels in the range of 5-10%, while for cheddar cheese, the salt was added to the curd after draining the whey to a level of 2%. Table I describes the Domiati cheese yield of control and contaminated samples. The cheese yield was increased by salt concentration and addition of pesticide. The curd from the contaminated milk had a softer and weaker texture than the control (2(5).



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13. MORSY ET AL.

Figure 5. Inhibition curve for dieldrin in cheddar cheese whey.


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Table L Cheese yield of control and contaminated samples at 0-10% salt. Cheese yield (%) Salt cone. (%)

Control samples


0.0 2.5 5.0 7.5 10.0

Contaminated Samples 20.34 28.22 33.22 33.41 37.02

18.80 23.90 28.42 30.70 31.00

The effect of salt concentration on dieldrin in salted whey is shown in Table Π. The results clearly show that the content of dieldrin in whey was increased. The highest value of pesticide at 10% salt concentration was 0.75 ppm while it was 0.1 ppm at 2.5% salt concentration.

Table Π. The effect of salt concentration on dieldrin in whey. Salt cone. (%)

Dieldrin (ppm)

0.0 2.5 5.0 7.5 10.0

0.00 0.10 0.15 0.50 0.75

Table ΙΠ shows no significant differences in the concentration of dieldrin recovered from cheese extracts. Even with an increase in salt concentrations no dieldrin was detected in the extract. Lack of recovery is probably due to the poor solubility of dieldrin in water (27,28).

Table U L Aldrin/dieldrin concentration in domiati cheese extract Salt cone. (%) 0.0 2.5 5.0 7.5 10.0

Dieldrin (ppm.) < < < <

Immunoassays for Residue Analysis - ACS Publications - American

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