Prion protein in milk

Abstract

Background: Prions are known to cause transmissible spongiform encephalopathies (TSE) after accumulation in the central nervous system. There is increasing evidence that prions are also present in body fluids and that prion infection by blood transmission is possible. The low concentration of the proteinaceous agent in body fluids and its long incubation time complicate epidemiologic analysis and estimation of spreading and thus the risk of human infection. This situation is particularly unsatisfactory for food and pharmaceutical industries, given the lack of sensitive tools for monitoring the infectious agent.

Methodology/principal findings: We have developed an adsorption matrix, Alicon PrioTrap, which binds with high affinity and specificity to prion proteins. Thus we were able to identify prion protein (PrP(C))--the precursor of prions (PrP(Sc))--in milk from humans, cows, sheep, and goats. The absolute amount of PrP(C) differs between the species (from microg/l range in sheep to ng/l range in human milk). PrP(C) is also found in homogenised and pasteurised off-the-shelf milk, and even ultrahigh temperature treatment only partially diminishes endogenous PrP(C) concentration.

Conclusions/significance: In view of a recent study showing evidence of prion replication occurring in the mammary gland of scrapie infected sheep suffering from mastitis, the appearance of PrP(C) in milk implies the possibility that milk of TSE-infected animals serves as source for PrP(Sc).

Figure 1. Detection of native PrP^C in milk of human and animals.

A volume of 10 ml fresh, ultra-high temperature treated (UHT) and pasteurised (Past) milk were enriched for PrP^C using Alicon PrioTrap® technology. Concentrated PrP^C was analyzed by Western Blotting using PrP−mab 8B4 (Alicon AG). The molecular weight markers are indicated. Recombinant bovine PrP(25−241) (Alicon AG) was used as a standard.

Figure 2. Specific binding of anti-PrP monoclonal antibodies to milk PrP^C.

Various anti-PrP monoclonal antibodies were used for detection of PrP^C in fresh cow milk. A Tau-1 protein-specific monoclonal antibody was used as a negative control. Recombinant bovine PrP(25−241) (Alicon AG) and recombinant Tau-1 protein (Chemicon International) were used as a standard.

Figure 3. Glycoform patterns of milk and brain PrP^C.

Alicon PrioTrap®-enriched PrP^C from cow milk and brain homogenate was treated with PNGase or PNGase/SDS before Western Blotting using PrP−mab 8B4.

Figure 4. Elimination of PrP^C from milk.

PrP^C from 10 ml fresh cow milk was detected as described in figure 1, followed by two consecutive PrP^C removal steps, where the milk supernatant was incubated with Alicon PrioTrap® filtration resin for 30 min before immunochemical detection. The total amount of milk protein was analyzed by Silver Stain analysis.