STUDENT INSTRUCTIONS

Soybeans are nutritional superstars by providing essential dietary amino and fatty acids. In addition, consumption of soy products have been shown to reduce cholesterol levels, and reduce risks of kidney and heart disease, osteoporosis, and possibly some cancers. However, soybeans are not a "complete" protein source for people and animals since soybeans lack the essential amino acid methionine (“essential” here means that animals cannot create methionine themselves). To rectify this deficiency, plant biotechnologists used methods of recombinant-DNA technology to insert a gene into a strain of soybeans to enable them to synthesize this missing amino acid.

The problem with this approach was that the original source of the gene for methionine, "2S albumin," came from Brazil nuts (Bertholletia excelsa). Many people are allergic to Brazil nuts and their reaction can range from severe rashes to anaphylactic shock. The study we focus on here by Nordlee et al. (1966) addresses this issue.

As a reminder of basic human immuno-biology, allergic or immediate hypersensitivity reactions are caused by binding of the antibody Immunoglobulin E (IgE) to the allergen (in this case, the methionine rich 2S albumin). This causes a series of reactions including the release of substances such as histamine, prostaglandins, and other compounds that produce the symptoms of an allergic reaction listed above. A glossary below lists some terms you may not be familiar with.

The purpose of the study by Nordlee et al. (1996) was to determine the extent to which the transgenic soybeans, containing the Brazil nut 2S albumin gene, caused allergic reactions similar to Brazil nuts.

Figure 1 is based on data from a radioallergosorbent test (RAST) in which a sample of blood from a potentially allergic person is checked for allergic sensitivity to specific substances. The approach used by the RAST is to determine whether specific IgE antibodies in serum drawn from sensitized subjects are able to recognize the protein of interest, in this case the 2S albumin from Brazil nuts.

In the standard radioallergosorbent test (RAST), the allergen, in this case 2S albumin extracted from Brazil nuts, is bound to an inert solid support, such as a tiny glass bead or some other microscopic solid. This is called the "solid" phase. Next, solutions of the "solid phase" of the allergen are incubated with blood serum from people who were allergic to Brazil nuts, but not to normal soybeans. During this incubation, serum IgE specific to the allergen (2S albumin) will bind to it. Following this, excess serum and non-allergen specific IgE are then washed away leaving behind only the serum IgE specifc for, and bound to, the "solid phase" allergens. Next, a radio-labeled anti-IgE antibody protein is added that will bind to the IgE molecules currently bound to the "solid phase" allergens creating a kind of molecular sandwich. A subsequent wash then removes all unbound radiolabeled anti-IgE proteins. Thus, by measuring the amount of radioactivity in the remaining sample, one can estimate the amount of allergen specific IgE in the original serum sample, and the degree of IgE binding affinity (i.e. the allergenicity) of the allergen.

Nordlee et al. (1996) used a slightly more elaborate procedure involving multiple RAST assays in sepatare trials on serial dilutions of the liquid protein extracts from Brazil nuts, transgenic and non-transgenic soybeans. Each of these are referred to as "inhibitor proteins" because they compete for the binding sites of IgE's that are already bound to the solid phase produced as described above. In each RAST assay, if the "inhibitor proteins" (either protein extracts from Brazil nuts, transgenic and non-transgenic soybeans) have a high affinity for the serum IgE, relative to the affinity between IgE and the solid phase Brazil nut 2S albumin, then there will a high rate of binding between the "inhibitor proteins" and the IgE. This will inhibit and/or displace the binding of the IgE to the solid phase Brazil nut 2S albumin - thus the "inhibitor" competes with the solid phase Brazil nut 2S albumin. When this happens, IgE will remain in solution and will be washed out before the label anti-IgE is added. This will decrease the amount of radioactivity in the remaining sample, and will result in a lower radiation count in the final step of the RAST assay (again, because everything not attached to the solid phase will be washed away). Since the counts are a relative measure of affinity, the authors calculated an index of "Inhibition of IgE binding (%)" as:

(counts without inhibitor protein - counts with inhibitor protein) * 100%
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(counts without inhibitor protein)

Discuss the following:

Turn to your neighbor and take 5 minutes to interpret the figure.

Take a moment to absorb the equation above. Run it through in your mind replacing the words "inhibitor protein" with "Brazil nut protein." With very little added Brazil nut protein, the counts should be as high as they can get since all of the solid phase should get labeled and counted. The index of "Inhibition of IgE binding (%)" (y-axis) should be low in this case because the numerator should be near zero.

However, with a lot of added Brazil nut protein, a lot of the IgE will be displaced from the solid phase (because the added Brazil nut protein would compete for the IgE with the solid phase Brazil nut protein) and washed away, and the counts should plummet. The index of "Inhibition of IgE binding (%)" (y-axis) should be quite high in this case because the numerator should be large. Note that with more added Brazil nut protein, the counts should plummet to zero as all of the IgE is displaced from the solid phase and washed away. This would result in a value of 100% for the index of "Inhibition of IgE binding (%)." Do you and your neighbor understand why?

After understanding exactly what the axes mean in Figure 1, now ask and discuss answers to the following:

1. What were the main research questions the scientists were asking?


2. What was the benefit of conducting this test to determine the allergenicity of trangenic soybeans?


3. Contrast the slopes of the 3 treatments; what conclusions can you draw about the relative allergenicity of the transgenic soybean?


4. Did Pioneer Hi-Bred International (Dupont) make the right decision to abandon this research program? What were some alternatives?


5. The ethical question of whether transgenic foods should be labeled is hotly debated. Can you assess the value or importance of this experiment within the context of bioethics? Should governments require labeling of genetically engineered foods?


6. According to statements made by FDA Commissioner Dr. J. D. Henney (Thompson 2000, see Background to Figure Set 1) "we have no scientific evidence to indicate that any of the new proteins introduced into food by biotechnology will cause allergies." Based on your understanding of Nordlee et al. (1996), is Dr. Henney correct? Explain your response.

Glossary of terms

Allergy- overreaction of the immune system to specific substances called allergens (such as pollen or bee stings) that in most people result no symptoms. Allergies often involve IgE (one of the 5 types of immunoglobulins produced by humans) antibodies.

Allergen- an antigen that produces allergic reactions by inducing formation of IgE.

Antibody- proteins, produced by the immune system, that recognize a foreign substance and starts a process of removal of the foreign material from the body.

Antigen- a substance that stimulates the production of an antibody (see Allergen)

Immune system- a system in mammals that recognizes and then eliminates or neutralizes foreign substances.

Immunoglobulin- a group of proteins active in the immune system that serve as antibodies. They work by binding to foreign antigens.

The Health On the Net Foundation: Allergy Glossary (http://hon.ch/Library/Theme/Allergy/Glossary/a.html)