Inhibitory Substances(1)

This section is adapted from two reports prepared by: Mark Mitchell (1995), Ontario Ministry of Agriculture, Food and Rural Affairs, Guelph, Ontario

Regulations

Most jurisdictions have regulations concerning the testing methods and limits of certain antibiotics in raw milk. The Milk Act of Ontario, Regulation 761, Section 52, Subsection, states:

"The milk of every producer shall be tested at least once a month for the presence of an inhibitor by an official method."

An official method is described in a separate inhibitor policy document which states:

The minimum sensitivity of an official method to test for the presence of an inhibitor under section 52 of Regulation 761 shall be:

  1. 0.01 international units of penicillin per millilitre of milk by the Standard Disc Assay (Bacillus stearothermophilus) procedure.
  2. 10 parts per billion sulfamethazine by the High Performance Liquid Chromatography (modified Smedley and Weber) procedure.

A concentration of .01 international units of penicillin per millilitre of milk is equivalent to 6 parts per billion (ppb). Note: 1 ppb is equivalent to a single penny in $10 million or one second in 32 years.

Detection Methods

It is beyond the scope of this manual to discuss any specific methods in detail. What follows is are brief descriptions of five types of inhibitor tests which are currently used in the dairy industry. For each category one or more brand name tests are listed to indicate possible choices. For cheese manufactures seeking assistance with inhibitor testing, there are many private labs which provide suitable services. In Ontario, a wide range of expertise and methodologies are available from Laboratory Services Division, University of Guelph.

Growth Inhibition Assays

Examples: Delvotest P, Delvotest SP, BR test, BR-AS test, Charm Farm, and the Disk Assay

This test format involves a standard culture of a test organism in an agar growth media, usually Bacillus stearothermophilus, that is inoculated with a milk sample and incubated for periods of up to several hours. If the milk contains sufficient concentrations of inhibitory substances the growth of the organism will be reduced or eliminated. The presence of an inhibitory substance is indicated by zones of inhibition or a change in colour of the media (pH and redox indicators).

The major disadvantages of these tests are that they are not very specific for identification purposes, have limited sensitivities to many antibiotics and take a long time before results are available. Growth inhibition tests are only able to classify residues into either the ß-lactam (penicillin like antibiotics) or other than ß-lactam antibiotic families. A further concern is that growth inhibition tests are subject to the effects of natural inhibitors (eg. lysozyme, lactoferrin, complement and defensins) which can be found in high levels in mastitic milk and may give false positive test results, particularly when used at the cow level. These effects can be minimized by heating individual cow samples at 82C for 2-3 minutes in a microwave oven or water bath before testing to destroy natural inhibitors and allow antibiotics which are more heat stable to remain.

The advantages of these tests are that they are cheap, easy to perform and have a very broad detection range.

Enzymatic Colorimetric Assays

Example: Penzyme Test for ß-lactams

The penzyme test is based on the inactivation of an enzyme by ß-lactam antibiotics. The enzyme (DD-carboxypeptidase or penicillin binding protein) is present in all bacteria and is involved in the synthesis of the bacterial cell wall. ß-lactam antibiotics will bind specifically with this enzyme and block it's activity, thus preventing the formation of the bacterial cell wall. This enzyme has been freeze dried and placed in sealed vials to which the milk sample is added. After addition of 0.2 ml (200 µl) of milk sample to the vial the sample is incubated for 5 minutes at 47C. During this time any ß-lactams present in the milk bind to the enzyme and inactivate a certain amount depending on the concentration present.

Reagent tablets specific for the enzyme (D-alanine peptide and D-amino acid oxidase) are then added to the milk sample and the sample is incubated at 47C for 15 minutes. During incubation any remaining active enzyme will react with the reagent added. The end product of the substrate and enzyme reaction (pyruvic acid and hydrogen peroxide) is measured by a redox colour indicator and the final colour is compared to a colour chart provided with the kit.

An orange colour (reduced) indicates a negative test result.

A yellow colour (oxidized) indicates a positive test result.

Microbial Receptor Assays:

Example: Charm II

This test uses bacterial cells (Bacillus stearothermophilus), which contain natural receptor sites on or within the cells for antibiotics, and radio labelled (C14 or H3) antibiotics. Milk sample is added to a freeze dried pellet of bacterial cells (binding reagent) in a test tube and the sample is mixed and incubated. During incubation any antibiotic present in the milk will bind to it's specific receptor site. Radio labelled antibiotic (tracer reagent) is then added and the sample is mixed and incubated. Unbound receptor sites on the bacterial cell will be bound by the radio labelled antibiotic. The sample is then centrifuged to collect the bacterial cells in the bottom of the test tube and the supernatant and butterfat is discarded. The bacterial cells are then resuspended and mixed in scintillation fluid. Binding is measured with a scintillation counter and compared to a positive and negative control. The more antibiotic present in the sample the lower the scintillation counts determined by the equipment.

Charm currently has test kits in this format for ß-lactams, macrolides, aminoglycosides and sulfonamides.

Immunoassays

Unlike other residue testing methods immunoassays are fast, sensitive, inexpensive, reproducible, reliable and simple to perform. The technique depends upon the measurement of the highly specific binding between antibodies (Ab) and antigens (Ag). Antigens are substances which are foreign to the body (eg. bacteria, viruses, toxins, pollens, drugs, hormones and pesticides) and that when introduced into the body give rise to the production of antibodies. Antibodies are proteins produced in the body by white blood cells (lymphocytes) as a result of exposure to antigens (destroy invading pathogens). The extreme sensitivity of the immunoassay is due to the development of certain labelling techniques for molecules (conjugates), enabling the measurement of very small masses (picogram or parts per trillion) of substances.

Immunoassays are classified according to the label which is attached to either the antigen (the anolyte being measured) or the antibody. The label may be a radioactive atom as in radio immunoassays (RIA), or an enzyme as in enzyme immunoassays (EIA or ELISA (Enzyme- linked immunosorbant assay)) or a fluorescent substance as in fluorescence immunoassays (FIA).

There are 3 major types of immunoassays used commonly for the detection of antibiotics in milk:

  1. Enzyme-Linked Immunoassay (eg. LacTek tests, SNAP for Tetracyclines, Single Step Block for SMZ)
  2. Enzyme-Linked Receptor Binding Assay (eg. SNAP for ß-lactams, Delvo-X-Press)
  3. Radio immunoassay (CHARM II for tetracyclines and chloramphenicol)