Purification of Immunoglobulin G by Ion-Exchange

Purification of Immunoglobulin G by Ion-Exchange Purification of Immunoglobulin G by Ion-Exchange Chromatography and Immunoelectropheresis William McTavish Joseph Zappa Introduction Immunoglobins or, Antibodies, are soluble proteins secreted from host differentiated plasma cells that target and eliminate specific antigens to protect the host from disease (Jakoby, 1971). There are five isotypes of immunoglobulin: IgM, IgD, IgA , IgE and IgG, with IgG being the most prominent antibody found in blood circulation of the host. The purification of specific antibodies has led to the development of techniques such as western blotting; where desired proteins can be targeted by monoclonal antibodies engineered for a specific affinity for that protein( Burnette, 1981). The basis of immunoglobulin purification can begin with a technique of â€Å"salting out†, used vastly for precipitating organic molecules and is the first step in protein purification (Tsutomu and Timasheff, 1984). Immunoglobins are small soluble proteins that can be found within serum that is removed from a blood sample taken from the host. Hydrophillic immunoglobins contain amino acids that are polar or possess an ionic charge. Counter ions in the serum of the host are attracted to these polar and ionic charges making the proteins soluble in the solvent. By destabilizing the intermolecular forces between the immunoglobins and the serum solvent there can be an induced precipitation of these proteins. Ammonium sulfate is a highly used compound in salting out procedures, for when ammonium sulfate dissociates, the large sulfate ions form hydrogen bonds between the polar molecules found in the serum (Tsutomu and Timasheff, 1984). The quenching effect of sulfate removes hydrogen bonds and intermolecular forces away from the protein molecules, forcing them to form bonds between one another. This forced intermolecular bonding between proteins causes an accumulation of aggregated proteins and eventually, at the right concentration of salt, precipitation out of solution (Tsutomu and Timasheff, 1984). Although the precipitation of immunoglobulin from host serum with Ammonium sulfate is an efficient procedure for isolating globin, it does not allow for the accurate determination of a specific isotype of immunoglobulin. Ion exchange chromatography is a prominent technique used to acquire a single desired protein, including a specific isotype of immunoglobins. All molecules, including immunoglobulin that have ionizable groups have a net surface charge that is highly dependent on the environmental pH in which that molecule is in. The pH of an environment can dictate the amount of charge present on a molecule, whether it is more positive or more negative, as well as neutral (Grodzki, and Berenstein, 2010). The neutral point, where all positive charges cancel out the negatives is expressed as the pI of the molecule (Grodzki, and Berenstein, 2010). Since all proteins vary in their pI they will express specific charges at any specific pH. This characteristic of immunoglobulin is utilized in Ion exchange chromatography to isolate specific isotypes even if they vary only slightly in charge. IgG, as well as other isotypes of Ig, have a pI occurring near neutral pH so Anion exchange resins are often used for this type of chromatography. Anion Exchangers utilize resin that contains positively charged functional groups that act as counter ions towards protein being eluted through the column (Determann et al. 1969). With the resin set at a specific pH, the proteins that are most positive will exit the column first due to the repulsion of charges between the positive protein and positive resin. The next proteins to elute will be the neutral ones followed by the negatively charged proteins. Proteins are removed in this manner by constantly adding more of the buffer the column is immersed in. By adding more buffer there is an increased competition for associating with the resins charges, which in turn dissociates protein from the resin and further elutes them through the column (Determann et al. 1969). Not only does the charge of the beads matter but also the flow and porosity of the resin, alternations of these can allow for either a more broaden column exchange or a far more refined one. Diethyl aminoethyl (DEAE)-cellul ose is a commonly used resin for anion exchanging due to its higher porosity and positive functional groups that allows for better flow properties of the column. Increased flow rate allows for separation of more bulky and crude proteins, such as crude immunoglobulin, and aids in a higher resolution of separated proteins (Determann et al. 1969). Once several fractions of the column elution is collected there is many ways to identify which fraction is most likely containing the desired protein of isolation including determining the optical density of the fractions with a spectrophotometer. The OD of Immunoglobulin and other proteins can be determined by selecting a specific wavelength of light and beaming it through the elution fraction and recording the amount of transmitted light via photoreceptors (Edelhoch, 1967). A common wavelength used for identifying immunoglobulin is 280nm, this wavelength is absorbed by the amino acid tryptophan in proteins. Absorption of this wavelength in protiens makes it a proportional reduction of transmitted light based on the concentration of protein present in the column fraction (Edelhoch, 1967). The higher the reduction in transmitted light, the higher the OD reading for a fraction. A fraction of elute from Ion exchange chromatography may contain the desired Immunoglobulin G, but to further prove this, a technique called Immunoelectrophoresis (IEP) can be used to confirm the purity of Immunoglobulin fraction. Immunoelectrophoresis is a two-part technique that combines the use of electrophoresis and zone of equivalence of immune complexes to determine a positive result. Electrophoresis is another basic technique used in separating proteins based on size and charge to obtain separate sections of protein in agar gel or other resins such as polyacrylamide in SDS-PAGE techniques.(1) Proteins separate into a gradient of smallest more positive charged towards the cathode to smallest most negative charged towards the anode, with the larger, less charged proteins in the middle gradient. (Serwer and Wright, 2012). After protein separation has occurred in the welled samples, there is addition of antibody specific for certain protein that may be isolated out of the samples used in the experiment. If proteins are present that are the target of affinity for the added antibodies there will be association of antibody:antigen complexes. These complexes will form in the agar gel and at the proper gradient of both antibody and antigen concentrations there will be precipitation of these complexes out of the solution (Slater, 1975). This correct gradient is called the zone of equivalence and is frequently used in determining the presence of desired protein molecules, including immunoglobulin (Slater, 1975). Several other techniques are used in isolating proteins, an extremely prominent technique is the use of Antibodies themselves in Immunofluorescence (IF). Antibodies are engineered to contain a specific affinity towards a desired molecule, protein or even a whole cell. IF can work in either two ways: the first involves a single antibody engineered towards a desired antigen containing a flurochrome itself and emits fluorescent light to be detected. The second contains a secondary antibody that has affinity for the primary antibody binding to an antigen, this secondary antibody is the one that contains the fluochrome for detection (Johnson, 2006). In either of these techniques there is the advantage of staining samples of proteins or cells and identifying not just a single antigen but several with several different antibodies. This technique is extremely useful for identifying proteins in cell structures as well as identifying the presence of proteins in biological systems. Methods and Materials All methods used in this experiment can be located in the Immunology Laboratory Manual cited in references. There were two major alternations to the Immunoelectrophoresis experiment; There was a time of 1.5 to 2 hours allowed for electrophoresis of the agar slides instead of 1 to 1.5 hours. There was also an expansion of time from 24 hours to 48 hours allowed for the IEP slide to rest in a cold room before soaking in 1% NaCl solution. Results A high concentration of IgG was isolated in the third elution fraction from DEAE-cellulose Ion exchange chromatography. Optical density of six Ion exchange chromatography elution fractions were taken with a spectrophotometer to determine protein concentration at a wavelength of 280nm (Fig 1). The highest optical density was observed in the third elution fraction (Fig 1). This illustrates that the largest concentration of protein at a similar charge was eluted at the third fraction of the Ion exchange experiment. Figure 1. Third fraction of DEAE-celluose elution scored the highest optical density. All fractions were tested with spectrophotometry and optical density measurements were taken at a wavelength of 280nm (Fig 1). Results are shown as single values of optical density (OD) and relate to the amount of protein concentration in each fraction. (Fig 1) Immunoelectrophoresis of isolated protein reveals presence of purified IgG in response to Goat anti-rabbit serum Presence of Rabbit Immunoglobin was tested for using Immunoelectrophoresis with Goat anti-rabbit serum. Normal rabbit serum and purified fraction of protein were welled on a 1% agar slide and proteins were separated based on charge via electrophoresis. Anti-rabbit serum was added and results were taken for precipitation of immune complexes 48 hours later (Fig 2). Thin white lines between the wells and trough are precipitated immune complexes and thus show a positive test for rabbit immunglobins (Fig 2). Figure 2. Precipitated immune complexes reveal immunoglobin presence in normal rabbit serum and purified fraction. Proteins were isolated based on charge via electrophoresis to isolate specific proteins. Goat Anti-rabbit serum was added as antibody for rabbit immunoglobin and incubated for 48 hours. Distinction of grey and white bands are positive results regardless Discussion Purified Rabbit Immunoglobin G was isolated from Normal rabbit serum using DEAE-cellulose ion exchange chromatography and Immunelectropheresis with Goat anti-rabbit serum. Once the majority of proteins were salted out of the normal rabbit serum, Ion exchange chromatography was used to separate all proteins from the sample of crude globin. Since immunoglobin proteins are soluble in the blood and are near neutrally charged at philological pH, a large amount of protein was expected to elute roughly half way through the Ion exchange chromatography regardless of using anion or cation exchange columns (Grodzki and Berenstein, 2010). These results occurred for the DEAE-cellulose Ion exchange column used to separate crude rabbit globulin in our experiment. The third elution fraction, of six, contained the highest optical density when evaluated with the spectrophotometer at 280nm. Optical density is related to the concentration of protein in a sample, thus the fraction containing the highest amount of protein was the third fraction which was collected half way through the elution process. Although the method of determining sample concentrations for proteins can vary, these results can be seen in similar protein isolation studies such as Ye et al. article Isolation of lactoperoxidase, lactoferrin, ÃŽ ±-lactalbumin, ÃŽ ²-lactoglobulin B and ÃŽ ²-lactoglobulin A from bovine rennet whey using ion exchange chromatography. The protein isolated is presumed to be the globulin isotype Iummogloublin G, this is due to the nature of circulating antibodies found in the serum of the rabbit. The most prominent antibody isotype circulating in the blood is IgG, which binds to antigens, forming immune complexes as well as aiding in many other immune system mechanisms such as compliment activation, opsonization and etc (Collins and Jackson, 2013). Immunoelectrophoersis with Goat anti-rabbit serum was used next to determine whether or not the isolated protein in the third elution fraction is Immunoglobulin G. The nature of this experiment depends on two key process gel electrophoresis and precipitation of Immune complexes. If electrophoresis is preformed properly there should be a separation of proteins based on charge/size from the samples that were welled on the agar covered slide used in the experiment; creating small zones of protein purity along the slide (Slater, 1975). Since the eluted fraction sample should only contain one kind of protein and is roughly pure, there should only be one zone of protein sample, where the normal rabbit serum, containing an array of different proteins, will electrophoresis out into several different zones of protein. Determining these zones of protein was done by adding Goat anti-rabbit serum and allowing diffusion into the gel to create zones of equivalence between antibody and antigen, thu s precipitating the complex to be seen visibly (Serwer and Wright, 2012). For a positive result on the purity of the fraction sample only a single precipitation line formed at the zone of equivalence would be seen. The results for the purity of the fraction sample was conclusive with the above expectations, only a single faint precipitated line was seen on the gel; therefore re-enforcing that there is only a single protein isolated from the Ion exchange elution phase. The single protein isolated is promptly IgG due to it’s response to the anti-Rabbit serum containing anti-rabbit globulin. Immunelectrophoresis was used in this experiment to confirm the presence of IgG in the eluted fraction sample taken from DEAE-cellulose ion exchange chromatography. The reason this method was used was due it’s simplicity in determining specific immune complexes and thus re-ensuring purity. It is relatively quick in determining the presence of antigen, in this case the immunoglobin G of rabbit, and gives results ready to be read visually, lacking the need for software or other means of identification. The draw back of this technique is that it takes some practical skill in preparation and is only useful in identifying the purity of one sample at a time. Techniques such as western blotting would be more efficient for studies that desire more than a single purity such as Yang et al’s article Correlation between the overexpression of epidermal growth factor receptor and mesenchymal markers in endometrial carcinoma. An alternation to this experiment could be made in the chroma Purification of Immunoglobins is an extremely useful procedure. Being able to isolate specific classes of Immunoglobulin aids in research of host immune deficiencies such as the research done by Tamura et al in their article Tumor-Produced Secreted Form of Binding of Immunoglobulin Protein Elicits Antigen-Specific Tumor Immunity as well as many other fields of host immunity and clinical research. Successful purification and crystallization of Immunoglobulin has also allowed for insight on how host immune systems respond to infection and the biological processes that take place in these responses. References Jakoby, W.B. 1971. Cystallization as a purification technique, Enzyme Purification and Related Techniques, Methods in Enzymology. 22: 246-252 Determann, H. Meyer, N. Wieland, T. 1969. Ion exchanger from pearl-shaped cellulose gel. Nature 223: 499-500 Edelhoch, H. 1967. Spectrospoic determination of tryptophan and tyrosine in proteins Burnette, N.W. 1981. â€Å"Western Blotting†: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochem 112: 1935-203 Tsutomu A. and Timasheff, S.N. 1984. Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding Biochemistry(23)25:5912-5926 321 -Grodzki, A.C. Berenstein, E. (2010) Antibody Purification: Ion-Exchange Chromatography Methods in Molecular Biology 588: 27-32 Slater, L. 1975. IgG, IgA and IgM by formylated rocket immunoelectrophoresis. Ann Clin Biochem 12 (1) : 19-22, 24 Yang, W.N.Ai, Z.H. Wang, Xu, J.Y.L. Teng, Y.C. 2014.Correlation between the overexpression of epidermal growth factor receptor and mesenchymal makers in endometrial carcinoma. J Gynecol Oncol. 25:36-42. 47 Collins, A.M. Jackson, K.J.L. 2013. A temporal model of human IgE and IgG antibody function. Front Immunol 4: 225 Ye, X. Yoshida, S. Ng, T.B. 2000. Isolation of lactoperoxidase, lactoferrin, ÃŽ ±-lactalbumin, ÃŽ ²-lactoglobulin B and ÃŽ ²-lactoglobulin A from bovine rennet whey using ion exchange chromatography The international journal of Biochemistry Cell biology 32 (11-12): 1143-1150 22 Nydegger, U.E. Lambert, P.H. Gerber, H. Miescher, P.A. 1974. Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq Circulating immune complexes in the serum in Systemic Lupus Erythematosus and in carriers of Hepatitis Antigen B Quantitation by binding to Radiolabelled Clq. J Clin Invest.  54(2): 297–309. Serwer, P. Wright, E.T. 2012. Agarose Gel Electrophoresis Reveals Structural Fluidity of Phage T3 DNA Packaging Intermediate. Electrophoresis 33 (2): 352-365 101-Johnson, I.D. 2006. Practical considerations in the selection and application of fluorescent probes. In: Handbook of biological confocal microscopy, 3rd ed. (J.B. Pawley.ed), Plenum Press. new York. p.362-3. Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq

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