To study a simple, and large-scale separation method of IgG from immunized hen egg.
Antibodies widely available for the scientific and diagnostic purposes are mainly mammalian monoclonal and polyclonal antibodies. Smaller animals such as mice and rats were used for the production of monoclonal antibodies while horses, sheep, pigs, rabbits and guinea pigs were used for the production of polyclonal antibodies. The major disadvantage of monoclonal antibody production is that some of the antigens are weakly immunogenic for mice. In polyclonal antibody production, the purification of the antibodies of interest from the mammalian blood was found to more difficult and labour intensive. Antibodies can be harvested from the egg yolk of birds instead of serum, which possess a wide range of application in antibody production. Also the antibody productivity of egg-lying hen is greater when compared to similar sized mammal. Thus the hens are thought as a convenient and cheap source for the production of antibodies.
Klemperer, in 1893, first demonstrated the immunization of a hen that resulted in the transfer of specific antibodies from the serum to the egg yolk. Egg yolk of immunized egg is a rich and inexpensive source of polyclonal antibodies. The use of laying hens for antibody production led to the reduction of animal use for antibody production. Also the collection of blood from animal is replaced by antibody extraction from egg yolk. A hen can lay egg continuously for about 72 weeks; then its capacity reduces gradually. Chickens produce unique antibodies called IgY. Chicken IgY antibodies isolated from egg yolk are the equivalent to mammalian IgG antibodies. A single egg yolk from an immunized chicken contains approximately 300mg of IgG antibodies. Whole eggs or separated egg yolks can be collected and stored frozen for later extraction of antibodies. Antibodies extracting from egg yolk are simpler to produce in larger amounts whereas other polyclonal and monoclonal antibodies are expensive and difficult to produce in a large scale ratio.
Moreover, chicken antibodies are thought to have many advantages over traditional mammalian antibodies. It differs from the mammalian IgG antibodies mainly regarding to their functions. The structure of chicken IgG antibodies is in such a way to interact with many epitopes of the mammalian antigens. The structure of chicken IgG antibodies is in such a way to interact with many epitopes (definition) of the mammalian antigens. IgY can be used in most biochemical and cellular applications, including western blots. The Fc region of chicken IgY is sufficiently different from mammalian IgG, so that chicken antibodies do not cross-react with mammalian proteins; also do not bind with mammalian rheumatoid factors, Fc-receptors or with proteins A or G. Therefore the background signal or false response in certain immunochemical assays is reduced. Generally the structure of IgY is similar to that of IgG with 2 heavy chains with a molecular mass of about 67-70 KDa each and two light (L) chains with the molecular mass of 25 KDa. The noticeable difference is regarding the number of constant regions (C) in heavy chains. IgG structure consists of 3 C regions (Cv1 – Cv3), while IgY has 4 C regions (Cv1 – Cv4). The additional C region in IgY corresponds to carbohydrate chains contributes to the greater molecular mass of IgY (180KDa) compared to IgG (150 KDa). The absence of the hinge between Cv1 and Cv2 makes IgY less flexible than IgG. Near the boundaries of Cv1–Cv2 and Cv2–Cv3, IgY is composed of proline and glycine residues enabling only limited flexibility. Half-life time of IgY is in months and that they retain their activity for 6 months at room temperature or for one month at 37°C.
Chicken antibodies can be prepared in large scale range. Chicken egg yolk antibodies will play an increasing role in research, diagnostics and immunotherapy in future studies. Recent research showed that purified chicken egg yolk antibodies will act as promising alternative for mammalian antibodies.
Extraction and Purification of Antibodies from Egg Yolk:
The entire extraction procedure should be carried out at room temperature. Seperate the yolk from the white and the cut open the yolk membrane. Pour the yolk and dilute with Tris buffered saline (TBS). Store the diluted yolk for prolonged periods in the cold. Separate the precipitate by centrifugation, allowing a reduction of amount of dextran sulphate necessary for next step. Mix the Dextran sulphatewith the diluted yolk after which add calcium chloride is added to induce precipitation of excess dextran sulphate. Wash the sediment with TBS to extract protein present in the solution. The first supernatant will be cloudy and the subsequent salt precipitation impeded if too little dextran sulphate and calcium chloride is added. Purify the IgG in the supernatant and concentrate it using sodium sulphate precipitation. Avoid cooling in this step as this will induce crystallization of sodium sulphate. Dissolve the precipitate is dissolved in a convenient volume of TBS for the future purposes.
Transfer the pooled IgG fraction obtained from egg yolk to an activated dialysis bag. Carry out the activation process is carried out by boiling the dialysis bag for 10 minutes; tie the dialysis bag, twist the open end of the dialysis bag to develop reasonable pressure. Suspend the dialysis bag in phosphate buffer (pH=7.2). Once the dialysis was over the gamma globulins separated can be then used for further purification using Ion Exchange Chromatography.
Separation of Immunoglobulins by DEAE Chromatography:
Ion exchange chromatography is one of the most frequently used chromatographic techniques used for the separation and purification of biomolecules such as proteins, polypeptides, nucleic acids, polynucleotide and other charged biological molecules. It has widespread applications in research because of its high resolving power, its high capacity and the simplicity and other control capabilities of the method. Common ion exchangers include DEAE, diethylaminoethyl (a positively charged anion exchanger) and CM, carboxymethyl, (a negatively charged cation exchanger).
Ion exchange chromatography basically involves two primary steps:
- Binding of a protein to a charged resin.
- Elution of the protein from the charges of the resin.
In order to prepare the DEAE cellulose column, add 2 to 3 g of DEAE cellulose to the beaker containing 500 ml of distilled water, stirr it well and keep it at room temperature for three hours with intermittent stirring. Discard the supernatant and treat the cellulose with 0.1N sodium hydroxide. Stirr it well and keep at room temperature for 30 minutes. Discard the supernatant , wash it with distilled water until a neutral reaction is observed. Add 0.1 HCl to the solution, again stirr it well and then allow to stand for 20-30 minutes. Discard the supernatant then wash it with distilled water, until there is a neutral reaction. Use the DEAE in the ratio of 5g for fractioning 1 ml of egg yolk fraction. The DEAE slurry is especially used for the fractionation of “IgG”.
Packing of Chromatography Column:
The fractionation of IgG from the egg yolk fractions needs a 30 ml glass burette which has been used as a column. Pack the column by first making the bed of the column with glass wool evenly, and then attach the tip of the burette to a rubber tube with pinch-cock arrangement. Fix the column fix to stand in the vertical position. Pour the DEAE slurry into the column, taking care not to allow formation of air bubbles and also to have an even distribution of the DEAE slurry and allow settling for some time. Once the column is set, wash the columnseveral time with 10 mM phosphate buffer with two gradients of 50Mm and 150Mm , saline (pH=7.4) till the outflow of buffer through the rubber tube arrangement showed the same pH 7.4 the fraction of flow rate was adjusted to 1 ml in 2 minutes. After packing the column, close the pinch cock and the egg yolk globulin fraction will layer on the top of the DEAE column. Once constant globulin fraction entered the matrix, maintain a continuous constant flow of more buffer and collect the fraction in 5 ml quantities. Maintain continuous flow of the buffer and collect the eluted fractions , check it for the IgG content using the Spectrophotometer at 280 nm. Pool and concentrate the fractions with 0.5 reading for future studies.
Concentration of IgG Fraction:
Transfer the pooled IgG fraction to a dialysis bag and tie it after including some air and twist the open end of the dialysis bag to develop reasonable pressure. Place it in a was petridish and cover it with finely powdered polyvinyl pyrorolidone (PVP) at room temperature. Tilt the petridish to keep the bag on higher side and to allow the fluid to come out of bag, to facilitate the flow. Water and other low molecular weight material will oozed out of the bag and form a viscous solution of PVP. To maintain the initial rapid rate of flow of liquid, squeeze the dialysis bag by twisting from one end and tied from time to time.
Chicken Egg Yolk Antibodies in Research:
- Immunized hen eggs are convenient and inexpensive sources of IgG antibodies.
- Amounts of antibody corresponding to almost half a liter of antiserum may be recovered from chicken in one month.
- Immunology has benefited from the easy dissection between the birds and mammals which have made the chicken the antibody products of choice when cross-reacting antibodies against mammalian antigens are desired.
- Studies shown that the IgG content of the egg is actually higher than that of hen’s serum.
- Antibodies extracted from hens are highly specific with stable titres up to 1:1,000,000.
- Chicken eggs are able to produce more specific antibodies against highly conserved mammalian antigens in contrast to rabbits.
- Wide application in medicine for therapeutic and prophylactic purposes.
- Purified antibodies can directly probe a specific antigen in Western blotting, ELISA and other applications.