Detection of Bovine Milk in Ovine Milk by an Indirect Enzyme-Linked Immunosorbent Assay

cation of specific components of milk fat or milk proteins include GLC (10, 20); gel electrophoresis (6, 9, 15, 17); isoelectrofocusing (12,21), and HPLC (3). Although these meth~ ods have their own individual merits, they are relatively costly, time-consuming,and require technical expertise. Immunological methods used for milk species analysis include agar-gel immunodiffusion (5) and its "dry disc" modification (7), radial immunodiffusion (13), rocket immunoelectrophoresis (18), hemagglutination inhibition (14), and inununodotting (1). All these methods use polyclonal antibodies raised against whey ~r casein proteins. Although accurate and sensItive the methods are time consuming and requke a high concentration of the antise~. Enzyme-linked immunosorbent assa~ l~ a rapid method that might have broad application for measuring components of foods that are capable of acting as antigens (4). Immunoassays differ from other modem methods of analysis in that the high technology is built into the reagents rather than the apparatus. Thus, specific and reliable analysis of trace components is possible without expensive apparatus or highly skilled operators. In this report, an indirect ELISA and blocked biotinylated antibodies against bovine whey proteins (BWP) were used to detect and quantify the presence of bovine milk in sheep's milk.
Detection of Bovine Milk in Ovine Milk by an Indirect Enzyme-Linked Immunosorbent Assay
TERESA GARCiA ROSARIO MARTiN, ELENA ROD.RIGUEZ, PALOMA MORALES, , PABLO E. HERNANDEZ, AND BERNABE SANZ Departamento de Higiene y Tecnologia de los Allmentos Facultad de Veterlnaria Universldad CompIutense 28040 Madrid, Spain ABSTRACT
Production of Polyclonal Antibodies
dissolved to 1 mg/ml in dimethylsulfoxide (DMSO, Merck), was added at a molar excess of 50 to the affinity-purified antibodies (antiBWP) dissolved in PBS (1.25 mg/ml, pH 7.2) and incubated for 2 h at room temperature (22). Unbound reagent was separated from the antibodies by overnight dialysis against PBS. Aliquots of biotinylated antibodies (.1 ml) were stored at -20·C until use.
INTRODUCTION
In most countries where ovine milk is produced either for direct consumption or for cheese making, it commands a considerably higher price than bovine milk. For economic as well as ethical reasoscertain that ovine milk offered for sale is free from admixtures with bovine milk. Several methods are available for identification of milk from closely related species. Nonimmunological methods, which require identifi-
1990 1 Dairy Sci 13:1489-1493
1489
1490
GARCIA ET AL.
centrifugation at 5800 x g for 10 min to remove precipitated caseins. The pH of the supernatant was adjusted to 7.0 with 1 M of NaOH and centrifuged at 5800 x g for 20 min to precipitate any remaining caseins. Supernatant containing the whey proteins was dialyzed overnight against phosphate-buffered saline (pBS, pH 7.2, containing .02%, wt/vol, sodium azide) and lyophilized. Dried whey proteins were placed in an airtight container and stored at -20·C until use. Antigenic whey protein extracts from ovine and caprine milk were prepared also.
Blocking of the Blotlnylated Antibodies
Polyclonal antibodies against BWP were raised in New Zealand male white rabbits. Immunization commenced with subcutaneous injection of lyophilized BWP (1 mg) emulsified in 1 ml of Freund's Complete Adjuvant (Difco Laboratories, Detroit, Ml) and 1 ml of deionized and distilled water. Multiple injection sites along the back were used. Booster doses (n 10) made in Freund's Incomplete Adjuvant were applied subcutaneously every 6 d. After 60 d, rabbits were bled. Blood was allowed to clot for 1 h at room temperature before serum was collected by centrifugation at 2000 x g for 10 min. Aliquots (1 ml) of the antiserum were stored at -20·C. Antibodies against BWP (anti-BWP) were recovered from whole antiserum by immunoadsorption (affinity) chromatography conducted at 4·C. Isolation of anti-BWP antibodies involved passage of 18-ml samples of diluted crude antiserum (1:2 with 2x concentrated PBST, pH 7.2 containing .5% Tween 80), through a CNBractivated Sepharose 4B column (Pharmacia Fine Chemicals, Uppsala, Sweden). Column contained 9 g of freeze-dried powder coupled to 158 mg of the lyophilized bovine whey proteins. Antibodies against BWP were released from the column by elution with .1 M glycineHCI buffer, pH 2.5. Eluted fractions with absorbance >.1 at 280 nm were pooled, adjusted to pH 7.2 with solid TRIS (Merck, Darmstadt, FRG), dialyzed overnight against PBS, and lyophilized prior to use in ELISA experiments.