Polyclonal antibodies (PAb) prepared against bovine milk alkaline phosphatase (ALP) were used to develop a competitive indirect (CI) ELISA. Anti-ALP PAb were specific for milk ALP and did not react with ALP from E. coli or bovine and calf intestinal mucosa. Anti-ALP PAb were 20% cross-reactive with bovine placenta ALP. The anti-ALP antibodies also did not recognize bovine serum albumin, acid glycoprotein, ovalbumin, ferritin, and casein, although some cross-reactivity was observed with whey protein isolate. Anti-ALP PAbs reacted with deglycosylated native ALP, but did not recognize ALP denatured at 100 degrees C in 2% SDS or deglycosylated denatured ALP. When buffered solutions of milk ALP were heated at 70 degrees C, ALP activity decreased at a faster rate than ALP content determined by CI-ELISA. The ELISA differentiated between native and heat denatured ALP. Further studies are warranted to determine if an ELISA can be used to verify pasteurization of fluid milk.

has been purified (Linden et al., 1974;Kazuya et al., 1980;Bingham et al., 1992), so in the production of polyclonal antibodies there is a need for the purification of sufficient quantities of the enzyme for immunization. Production of antibodies against bovine milk ALP could provide for the development of immunochemical assays that would determine adequate pasteurization of milk and differentiate bovine milk ALP from microbial ALP or ALP from other sources. The overall objectives of our study were to (1) develop a rapid method to purify sufficient quantities of ALP for polyclonal antibody production and (2) produce antibodies to bovine milk ALP and evaluate their cross-reactivity. MATERIALS & METHODS ElectrophoresisCrude bovine milk ALP (type XXI, EC 3.1.3.1), purified from skim milk, was obtained from Sigma Chemical Co. (St. Louis, MO). Initially, different electrophoretic buffer systems, with pH 4.5 to 10.2, were compared for optimal separation of ALP by native polyacrylamide gel electrophoresis (PAGE). In the final procedure, continuous native PAGE was performed in a Mini-protean II dual slab system (Bio-Rad, Hercules, CA) with an electrode buffer composed of 37 mM ammonia, 20 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS), pH 10.2. The resolving gel contained 6% acrylamide in 9.25 mM ammonia, 5 mM CAPS buffer, pH 10.2. For verification of purity, ALP was diluted to 10 mg protein/mL using sample buffer (3.7 mM ammonia, 2.0 mM CAPS pH 10.2, 20% glycerol and 0.2% bromophenol blue) and 30 L of this solution was applied to each well. For electrophoresis prior to electroelution, 40 L of ALP solution was used. The gel was run at a constant 100 volts for 40 min. The ALP band was located on the gel by incubating the native gel with an enzyme-specific stain containing 0.025% nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate in 0.1 M Tris buffer, pH 9.1. A black-purple band was observed when ALP activity was present.Sodium dodecyl-sulfate (SDS)-PAGE of ALP and milk proteins was performed using a Tris electrode buffer system (Laemmli, 1970). The resolving gel contained 12% acrylamide in 0.375M Tris buffer, 0.1% SDS, pH 8.8 and the stacking gel contained 4% acrylamide in 0.125M Tris buffer, 0.1% SDS, pH 6.8. ALP (or other proteins) was diluted to 10 mg protein/mL in denaturing sample buffer (0.0625M Tris, pH 6.8, 2% SDS, 10% glycerol, 5% mercaptoethanol and 0.2% bromophenol blue). The gel was run at a constant 200 volts for 45-50 min and stained with 0.1% w/v Coomassie blue (R250) dye in 50% v/v methanol and 10% v/v acetic acid or silver stain (Bio-Rad). Purification of ALP by electroelutionUnstained gel slices containing ALP were excised from the native gel by comparing to the relative mobility of the ALP band stained with NBT/BCIP. The gel slices were placed in the tubes of the Electro-Eluter (Model 421, Bio-Rad), and proteins were electroeluted using 20 mM CAPS buffer, pH 10.2, at a constant current of 8 mA/ tube within 3 to 3.5 h. The electroelution tank was placed in an ice ...