Allergy to cow milk in a test tube

Eveline Gouw, Honours student at the Institute for Life Sciences & Chemistry wrote a literature report about how allergic characteristics of food products (cow’s milk proteins) could be detected without the use of animals. This literature study was performed in collaboration with Bioceros and the research group Innovative Testing in Life Sciences & Chemistry.

Introduction

Nowadays, more and more people are developing allergies which calls for a more intense search for ways to treat them. Allergies develop because of environmental factors and genetic factors; the genetic variant is known as atopy, the predisposition to overproduce IgE (E. Hodge & I. Sayers, 2013). Cow milk allergy is the most common allergy in young children with about 3% affected, and often develops after starting with bottle feeding. When a baby is allergic to cow milk, his immune system reacts to proteins within the milk causing the digestive tract to inflame (Fitplein BV, 2014).

Food allergy is defined as a hyperactive immunologic reaction (hypersensitivity) to food and can be divided into IgE and non-IgE mediated reactions. Allergic reactions that causes the manifestation of acute symptoms by the individual after intake of the allergen are often listed under the IgE mediated reactions and have an effect on multiple organs: the skin (urticaria/angioedema), the respiratory system (asthma), the digestive system (pain/vomiting/diarrhea) and the cardiovascular system (anaphylactic shock). In IgE mediated reactions, crosslinking takes place between an allergen and the membrane bound IgEs of basophil and mast cells. When these cells are activated, granules containing inflammatory mediators are excreted. The optimal condition for excretion of these mediators depends on the concentration of allergen specific IgEs on the membrane, the concentration of allergens and the affinity of the IgEs for the allergen (K. Knipping et al., 2012).

In vitro degranulation assay

Researchers developed cow milk hydrolysates as a possible milk replacement for children with allergy to cow milk. The allergenic epitopes of the cow’s milk proteins are weakened in such way that they should not be able to cause an allergic reaction; this is done using hydrolysis.

Different test methods are available to prove that these cow milk hydrolysates indeed do not cause allergic reactions. The most used techniques for the analysis of peptides are HPLC, liquid chromatography-mass spectrometry, gel permeation chromatography and SDS-PAGE. To determine the antigenicity of the proteins, ELISA and Western Blotting methods are available and are also the most widely used. (K. Knipping et al., 2012).

Unfortunately, these test methods do not tell enough about how the cow milk hydrolysates will behave in vivo. Hence, before they can be tested in-vivo, more accurate in vitro methods are needed. For this reason, Bioseros has developed an in-vitro assay that tests for degranulation caused by allergens.

In this assay, rat basophils expressing human IgE receptors are used. These cells are exposed to the to-be-tested allergen, which in this case are cow milk hydrolysates. In order to get a reaction, crosslinking has to take place between the membrane bound IgE receptors and the IgE-allergen complexes.

The human IgEs that are necessary for this test vary too much because they are derived from different donors, and the availability is limited. As a result, the concentration of IgE in the serum is constantly different, causing the results of the in vitro assay to vary by a wide margin. Due to this limitation, a different antibody harvesting method was developed.

Obtaining the antibodies

To obtain the antibodies, mice are immunized with the allergen. This way, the body starts activating B cells which will be specialized for this allergen. The B cells are then isolated from the spleen and are fused with myeloma cells. Since the B cells are fused with myeloma cells, they will grow unrestrained and are still able to produce antibodies specific for the allergen. These fused cells are called hybridoma cells. The hybridoma cells are then grown in a way that they can be isolated per cell. This is necessary because every hybridoma cell originates from one specific B cell, which allows every single hybridoma cell to produce a different antibody. These antibodies will be purified later on and will consist of mostly IgGs.

Selection of antibodies and DNA synthesis

An antibody consists of two heavy and two light polypeptide chains which are held together by covalent and non-covalent bonds. Subsequently, every antibody consists of a constant and a variable part. The specificity of the antibody is determined by the variable part in combination with the binding of the heavy and light chains. Due to this variation, antibodies are very specific (M. Zouali, 2010)(Figure 1A). From the purified antibodies, about five antibodies will be selected for the assay. The selected antibodies have to be specific for one epitope of the allergen in order to have no crosslinking of the allergens. This is done to prevent competition between the antibodies later on (Figure 1B).

The DNA sequence of the selected antibody’s variable region, the part that binds the antibody to the allergen, has to be determined. The DNA sequence of the variable part of the mice IgG is then placed in front of the constant part of the human IgE DNA sequence. As a result, a chimeric antibody with a human antibody backbone is formed (Figure 1C). In front of the IgG-IgE sequence, a strong promoter is placed for optimal transcription. After the IgG-IgE sequence, a secretion signal is placed to let the cell secrete the antibody after transcription (Figure 1D).

Figure 1. A) Structure of an antibody. The heavy and light chains indicated on the left, and the constant and variable part indicated on the right. The covalent bonds are disulphide bonds (indicated with SS) and N-linked sugar groups. B) Schematic representation of an allergen to which different antibodies (orange, blue and purple) are bound. The purple antibody possesses a variable domain which is too similar to the variable domain of the blue antibody and therefore, will not be selected from all the obtained antibodies. C) Representation of a chimeric antibody with a human antibody backbone. The variable part (orange) is derived from the mouse IgG and the constant part (green) from the human IgE. D) Synthesized DNA with the strong promoter, the antibody sequence with the variable part of IgG and the constant part of IgE and a secretion signal in order to secrete the antibody (Figure: E. L. Gouw, 2013). The DNA sequence will then be placed into a vector. The vector will be transformed into Human Embryonal Kidney (HEK) cells which will transcribe the vector and produce chimeric antibodies. The secretion signal causes the antibodies to be secreted into the supernatant. The cells are spun down to purify the antibodies out of the supernatant so that they can be used for the in-vitro assay.

Examples of this technique

Some of the advantages of this technique are that the antibodies used for the in vitro assay are always the same antibodies and its availability is unlimited. This is because the antibodies originate from the same vector and can be synthesized over and over again. Since this new technique does not use human serum but synthesized serum, this assay has much less background degranulation. The human serum, used in the previous assays, contained many other compounds next to the antibodies, causing a very high background. This new method uses synthesized serum with only antibodies which provides a lower background. An unexpected but very practical advantage is that the assay is much more sensitive now that it is using chimeric antibodies, causing the results to be better than the previous method.

Possible applications

This newly developed technique is suitable for many different research directions. Examples include the study of other food allergies by using other commonly allergenic proteins instead of that from cow milk and make chimeric antibodies for these proteins (L.W. van den Elsen et al., 2013). This technique may also be suitable to look at the effectiveness of chimeric antibodies in anti-tumor therapy. In this area, much research has gone into using antibodies for binding site-specific antibody-drug conjugation for cancer therapy. The variable domain of an antibody from this technique, which would represent the tumor binding part, is fused with the constant domain which serves as a recognition site for potentially cytotoxic drugs. This chimeric antibody serve as recognition and binding sites for the medication, causing only the cancer cells to be exposed to the drug. IgG is the main type of antibody used for this application (S. Panowksi et al., 2014).

References

E. Hodge and I. Sayers (2013). Allergy. Encyclopedia if Life Sciences. John Wiley & Sons, Chichester. doi: 10.1002/9780470015902.a0001887.pub3

E. L. Gouw (2013). Figure 1 A-D.

Fitplein B.V. (2014). http://www.fitplein.nl/ideepagina/ideeen_popup.php?id=702. Accessed March 6, 2014.

K. Knipping, B. C. van Esch, A. G. van Ieperen-van Dijk, E. van Hoffen, T. van Baalen, L. M. Knippels, S. van der Heide, A. E. Dubois, J. Garssen and E. F. Knol (2012). Enzymatic Treatment of Whey Proteins in Cow’s Milk Results in Differential Inhibition of IgE-Mediated Mast Cell Activation Compared to T-Cell Activation. International Archives of Allergy and Immunology.159, 263-270. Doi: 10.1159/000338007

L. W. van den Elsen, L. A. Meulenbroek, B. C. van Esch, G. A. Hofman, L. Boon, J. Garssen and L. E. Willemsen (2013). CD25+ regulatory T cells transfer n-3 long chain polyunsaturated fatty acids-induced tolerance in mice allergic to cow's milk protein. European Journal of Allergy and Clinical Immunology. 68, 12, 1562-1570. Doi: 10.1111/all.12300

M. Zouali (2010). Antibodies. Encyclopedia of Life Sciences. John Wiley & Sons, Chichester. Doi: 10.1002/9780470015902.a0000906.pub2

S. Panowksi, S. Bhakta, H. Raab, P. Polakis and J. R. Junutula (2014). Site-specific antibody drug conjugates for cancer therapy. MAbs. 6, 1, 34-45. Doi: 10.4161/mabs.27022.