Volume 2: Science
5. Diagnosis and therapy
Detection of ruminant protein in animal feedstuffs
Enzyme linked immunosorbent assay for the detection of ruminant protein
Development of the ELISA

5.3 The ruminant feed ban came into operation in July 1988, and was the central element in the strategy to halt the BSE epidemic ¹ (see vol. 3: The Early Years, 1986-88 and vol. 5: Animal Health, 1989-96). However, in order to enforce the ban, a test was required which could detect ruminant protein and distinguish it from protein from other mammals and poultry present in cattle feed. The test developed to accomplish this was based on the ELISA technique. Enzyme linked immunosorbent assays are widely used in laboratories as a sensitive and cheap method to measure the quantity of a substance by using antibodies to that substance. In this instance, an ELISA was developed using antibodies that recognised heat-stable proteins (ie, proteins present in rendered material) derived from a variety of ruminant tissues and organs.

5.4 In the following sections, we give a brief explanation of the technical basis of this ELISA, a full account of which is given by Ansfield. ² We then give a brief description of the development of the test.

Enzyme linked immunosorbent assay for the detection of ruminant protein

5.5 In general, antibodies are produced by isolating antigenic fragments of the proteins of interest (ie, those parts which will induce the production of antibodies) and inoculating them into rabbits. The rabbits produce antibodies to these fragments which can subsequently be purified from their blood.

5.6 In the ELISA developed for the detection of ruminant protein, antibodies produced in this way are used to coat the surface of specialised plastic dishes. When suitably prepared samples of meat and bone meal (MBM) containing ruminant-derived protein are applied to these dishes, the specific antibodies recognise and bind the ruminant protein, but not protein from other species. The ruminant protein is therefore immobilised and bound to the plastic dish, and extraneous material can be washed away.

5.7 Further specific antibody is then added, although this time the antibody has been conjugated with an enzyme which catalyses the conversion of a colourless substrate to a coloured product. Thus, once the antibody has bound to the ruminant protein on the plastic dish, substrate is added and the enzymatic conversion of the substrate to a coloured product allowed to proceed. This is the detection step.

5.8 This method of carrying out an ELISA increases sensitivity as it incorporates an amplification step. This is because enzymes are capable of converting many molecules of substrate. Thus, one molecule of ruminant protein bound by one molecule of enzyme conjugate results in the conversion of many molecules of substrate.

5.9 The colour change in the substrate is proportional to the amount of ruminant protein in the sample. By testing known quantities of ruminant protein in parallel, it is possible to calculate the amount present in the MBM.

Development of the ELISA

5.10 Concern was first expressed in June 1988 by Mrs Elizabeth Owen (Food Standards Division, MAFF), that the ruminant feed ban would be unenforceable unless a test was developed that could distinguish between ruminant protein and protein from other mammals and poultry in cattle feed. ³

5.11 Mr Keith Meldrum (the Chief Veterinary Officer) invited comments on this matter in June 1988. ⁴ He received a reply from Dr Peter Dawson, his assistant, who was aware that staff at the Meat Research Institute of the Agricultural and Food Research Council were developing tests based on the ELISA to distinguish species differences in meat used in processed foods. ⁵ Following discussions between MAFF and the Meat Research Institute, it was agreed that a test based on the ELISA method would be useful for detecting ruminant protein in feedstuffs. It was also decided that the initial development of this ELISA test would involve detecting ruminant protein in rendered animal material before attempting detection in compound feedstuffs. ⁶ Work started on developing the test in March 1989, ⁷ but progress was hindered by a series of problems which included delays in the preparation of appropriate antibodies, ⁸ and the need to find methods of separating gelatine from the samples prior to testing, as gelatine solidification interfered with the test. ⁹

5.12 By December 1990, the initial test for detection of ruminant protein in rendered animal material had been validated. ¹⁰ The test was then developed to include porcine protein in late 1993, and the method was finally patented and reported in 1994. ¹¹

5.13 However, problems were encountered in applying this test to compound feedstuffs. These feedstuffs can contain protein from plant material and from various animal sources such as fishmeal and bloodmeal, not just rendered ruminant material. Several compound feedstuffs gave positive results even though there was no ruminant protein present in them. ¹² Subsequent improvement in protein extraction procedures minimised the problems associated with compound feedstuffs. ¹³

5.14 On-farm testing of cattle feed began in June 1994 on farms where Born After the ruminant feed Ban (BAB) cases had arisen. ¹⁴ By 1996, 1,577 samples had been tested, of which only three gave positive results. ¹⁵ It was thought that these results were due to low-level cross-contamination of ruminant feed by ruminant protein in pig and poultry feed. ¹⁶

5.15 In November 1994 the feeding of cattle with any mammalian protein was banned in an attempt to prevent spread of disease, and to comply with direction from the EU. ¹⁷ The potential for cross-contamination, along with the continued fear that the ruminant feed ban was not being adhered to, led the European Commission, in 1995, to introduce a more widespread monitoring programme to test for the inclusion of ruminant protein in ruminant diets using the ELISA test. ¹⁸

5.16 In March 1996, the UK Government banned the use of mammalian MBM in feed for all farm animals and prohibited the production of pet food on any premises where such animal feed was prepared in order to prevent cross-contamination. ¹⁹

5.17 Full validation of the ELISA test for compound feedstuffs was not obtained until 1997 because of the increasing range of feed ingredients and specific EU requirements. ²⁰ The results were published in 1998. ²¹ Delays in development of the test and the limited capacity of the MAFF laboratory to carry it out meant that it had little impact in monitoring the effectiveness of the ruminant feed ban. The development of the ELISA test for detection of ruminant protein in feed is discussed fully in vol. 5: Animal Health, 1989-96.