Skip to main content Accessibility help

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

  • Nathalie Geneix (a1), Eric Dufour (a2), Annie Venien (a1) and Didier Levieux (a1)


The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.


Corresponding author

*For correspondence; e-mail:


Hide All
Aschaffenburg, R & Mullen, JEC 1949 A rapid and simple phosphatase test for milk. Journal of Dairy Research 16 5867
Black, R, Kuzyk, M & Duggan, J 1992 Evaluation of a fluorometric assay for alkaline phophatase in fluid dairy products. Australian Journal of Dairy Technology 47 6467
Blel, M, Guingamp, MF, Gaillard, JL & Humbert, G 2002 Studies on the thermal sensitivity of γ-glutamyl transpeptidase measured with a modified test procedure and compared with that of alkaline phosphatase and lactoperoxidase in milk. Lait 82 555566
Chen, CC, Tai, YC, Shen, SC, Tu, YY, Wu, MC & Chang, HM 2006 Detection of alkaline phosphatase by competitive indirect ELISA using immunoglobulin in yolk (IgY) specific against bovine milk alkaline phosphatase. Food Chemistry 95 213220
Dobozy, K & Hammer, BW 1969 Some properties of alkaline phosphatase in Bacillus species. Acta Microbiologica of the Academy of Sciences Hungarian 16 181184
Glew, RH & Heath, EC 1971 Studies on the extracellular alkaline phosphatase of Micrococcus sadonesis. II: Factors affecting secretion. Journal of Biological Chemistry 246 15661574
Gorman, J & Hu, W 1969 The separation and partial characterization of L-histidinol phosphatase and an alkaline phosphatase of Saccharomyces cerevisiae. Journal of Biological Chemistry 244 16451654
Hammer, BW & Olson, HC 1941 Phosphatase production in dairy products by microorganisms. Journal of Milk and Food Technology 4 83101
Knight, AH & Fryer, SM 1989 The development of heat resistant phosphatase activity in raw milk. Journal of the Society of Dairy Technolology 42 81868193
Kumar, P, Sud, RK & Gupta, KG 1973 Interference of some pesticides in the milk phosphatase pasteurization test. Journal of Dairy Science 56 553557
Levieux, D, Geneix, N & Levieux, A 2007 Inactivation-denaturation kinetics of bovine milk alkaline phosphatase during mild heating as determined by using a monoclonal antibody-based immunoassay. Journal of Dairy Research 74 296301
Levieux, D & Venien, A 1994 Rapid, sensitive two-site ELISA for detection of cow's milk in goats' or ewes' milk using monoclonal antibodies. Journal of Dairy Research 61 9199
Manolkidis, KS & Alichanidis, ES 1971 Effects of some antibiotics on the milk phosphatase pasteurization test. Journal of Dairy Science 54 335338
Morton, RK 1953 Alkaline phosphatase of milk. 1. Association of the enzyme with a particulate lipoprotein complex. Biochemical Journal 57 786795
Murthy, GK & Cox, S 1988 Evaluation of APHA and AOAC methods for phosphatase in cheese. Journal of the Association of Official Analytical Chemists 71 11951199
Murthy, GK & Kaylor, LO 1990 Evaluation of APHA and AOAC II methods for phosphatase in butter and differentiation of milk and microbial phosphatases by agarose-gel electrophoresis. Journal of the Association of Official Analytical Chemists 73 681987
Rachetti, G, Fossati, G, Comitti, R, Putignano, S & Galante, YM 1987 Production of monoclonal antibodies to calcitonin and development of a two-site enzyme immunoassay. Molecular Immunology 24 11691176
Snow, LD 1994 Effect of n-butanol extraction on the pH optima of 5’-nucleotidase and alkaline phosphatase of milk fat globule membranes. Biochemical Archives 10 133139
Vega-Warner, AV, Wang, CH, Smith, DM & Ustunol, Z 1999 Milk alkaline phosphatase purification and production of polyclonal antibodies. Journal of Food Science 64 601605
Vega-Warner, AV, Gandhi, H, Smith, D & Ustunol, Z 2000 Polyclonal-antibody-based ELISA to detect milk alkaline phosphatase. Journal of Agriculture and Food Chemistry 48 20872091


Related content

Powered by UNSILO

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

  • Nathalie Geneix (a1), Eric Dufour (a2), Annie Venien (a1) and Didier Levieux (a1)


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed.