ImmunoAssay
Detection of Meat Adulteration

This page gives some theory about Immunology, precipitin reactions and the Ochterlony test. Procedures needed to test for the adulteration of cow, pig or horse meat follow. First, ensure that you have the materials and reagents needed for these assays. You can also jump to other lab procedures in an appendix, the biochemistry lab schedule, my home page or the addresses at the bottom. 

1. is highly specific (immunity to chicken pox does not protect against other viruses, even other pox viruses),
2. involves a form of cellular memory, since protection lasts for years, and
3. can distinguish between "self" and "non-self" since you normally don't respond to your own proteins, but do against another person's or animal's.

When an antigen is mixed with the correct concentration of its specific antibody, a precipitate forms. This is a latticework of antigen molecules bound together by antibody molecules, which each have two (divalent) or more (polyvalent) binding sites for a single part of the antigen's surface. Thus, each antibody can bind to two or more antigens at the same time. In addition, each antigen usually has several regions of unique structure (called antigenic determinants), each of which can induce production of a specific antibody to bind to it. Thus, each antibody preparation is usually a mixture of different antibodies, each specific for a different part of the antigen, as indicated in figure 1. below.

Also, the presence or absence and the amount of precipitate which results when mixing antigen with antibody (the precipitin reaction) is dependent on the relative concentrations of each. Thus, in a large excess of antibody, each will bind to only one antigen site at a time, on average, forming soluble complexes. At equivalence, each antibody molecule will tend to link the maximum number of antigens to produce the maximum precipitate possible. In large antigen excess, available antibody will be quickly bound to two (or the valence number of) antigens, such that small, soluble complexes again predominate (see Fig. 1.).

Figure 1. Antigen-Antibody Complexes

In 1948 S.D. Elek and O. Ouchterlony independently described a test based on the precipitin reaction, in which antigen and antibody diffuse toward each other from separate wells cut in agar. When they meet in optimal concentration, a precipitate forms in a band between the wells, but only if the antibody is specific for that antigen. Often the line is offset toward one well and curved rather than straight. This indicates that either the antigen (Ag) or antibody (Ab) has diffused more rapidly than the other. While a number of causes can produce this result, it is usually based on molecular size, with the smaller molecule producing a line curved away from and more distant from its own well (Figure 2.).

If there are multiple components in either preparation, multiple bands can form due to the different diffusion rates of molecules of different sizes. Thus, this test can be used to check purity. In addition, by placing different antigen preparations in neighboring wells equidistant from the antibody well, this assay can test for identity, partial identity, or nonidentity of the antigens with respect to the antibody used. If the same Ag is present in adjacent wells, a continuous precipitin line forms (Fig. 3.a). If the Ag mixtures contain the same Ag and also a different one, a spur forms (Fig. 3.b). Different Ags produce crossing lines (Fig. 3.c).

Thus, this or similar tests are extremely valuable for determining the purity of cell fractions and for indicating the identity or proving the lack of identity between two isolates. It should be stated that an identity pattern does not necessarily prove molecular identity, since different antigens may share the same antigenic determinant (partial surface shape) recognized by the antibody used.

Equipment Needed:

Reagents Needed:

1. Obtain a solid, 1.6% agarose plate and well cutter. Attach the tubing from the side arm of the vacuum flask to a source and sterilize the cutter with flame and/or 70% ethanol.
2. Using a gentle vacuum, cut the number and arrangement of wells into the agar sector(s) as needed using the template below (Fig. 4).

Keep the plate covered as much as possible to minimize contamination and be certain you have removed the cut agar plugs before trying to fill the wells.
3. Fill the wells with dyes, salt solutions or Ags and Abs using 10 or 20 µL Wiretrol^® capillary pipets.
For example, wells 2 and 6 might be filled with the dyes to demonstrate double diffusion, and then wells 4 and 8 filled with the salt solutions to show where BaSO₄ precipitates. These results can be seen after about 45 minutes to 1 hour.
4. To test control albumins (beef, pig and horse, for example) against the antibodies to ensure they are all active, one could put the Ags in wells 1, 5 and 9 or 3, 5 and 7, and the Abs in 2, 4, 6 and/or 8.
5. To test the effect of dilution on precipitin line shape and position, one could dilute the Ag (bovine albumin) by 1/1 to 1/10 or more with phosphate buffered saline (PBS) and fill wells 2 through 8, leaving the Ab in well 5. Alternatively, dilute the Ab and test against Ag in well 5.

Work quickly and be careful not to tip or jar any solutions out of the wells. You may be asked to use only 10 µLof each to conserve supplies (note this for your report).
6. To test your own meat for adulteration, bring about 5 grams (thumb-tip size) of raw, ground meat to the lab. Check the package label carefully for terms like "Ground Meat," or "Ground Beef," and record this for your report (these terms have specific definitions in USDA regulations).
7. Mash the sample in about 5 mL of PBS and drain the fluid into a vial to use as the Ag sample.
8. To test various meat extracts or "Mystery Meat" samples provided, one could fill wells 2, 5 and 8 with these and/or control Ags, and fill 3, 6 and 9 with the Abs and repeat the Abs in wells 7, 4 and 1. Thus, wells 3 and 7, 4 and 6, and 1 and 9 each contain the same Ab. This subjects all three Ags to all three Abs in one sector. Be aware that secondary interactions are probable with such multiple testing.

Figure 4. Template for cutting equidistant wells in agar plates

Questions to help analyze the results:

1. With which Ab did the standard Ag (bovine albumin) react? With which did it not react? How does this support or refute the idea of immune specificity?
2. Discuss the fact that a goat can produce Abs to serum albumins of other animals but not its own.
3. How might this test be used clinically, by the Red Cross faced with AIDS, etc. in the blood supply, and by the US Department of Agriculture?
4. Which protein is larger, the antigen or antibody? What evidence supports this?
5. What pattern (see Fig. 3) resulted between each set of wells? What does this mean?
6. Did dilution of the Ag or Ab affect the position or shape of the precipitin band? Explain.
7. Which animals were the source of each meat sample tested?
8. Antigen from which animals were not present in each sample?
9. Is there evidence of adulteration in any sample? Explain.
10. Could the meats be adulterated and still escape detection? How?
11. If a meat sample produced no precipitin line (did any?), what animals could not be the meat source?
12. Why is raw rather than cooked meat required for this test?

If you have questions or comments, write the:

  Author of this page: Terry Helser - helsertl@oneonta.edu
  Web Coordinator: Steve Maniscalco - maniscsj@oneonta.edu
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  Last Modified on 8/14/01