The Separation, Isolation, Purification and Characterisation of IgG from Non-Epileptic and Epileptic Patients Singh Vijender1,*, Khan Moinuddin2, Chauhan Manju3 1Ph.D Student, Department of Biotechnology, Singhania University, Jhunjhunu, Rajasthan, India 2Professor and VC, Department of Administration, Singhania University, Jhunjhunu, Rajasthan, India 3Professor and Head of Department of Bioscience, D.A.V. (PG) College, Muzaffar Nagar, Uttar Pradesh, India *Email id: vijendersingh2005@yahoo.co.in
Abstract Research work on the separation, isolation, purification and characterisation of IgG from non-epileptic and epileptic patients was carried out to examine the unexplored and overcome neurological disorders (epilepsy) with the help of various sophisticated techniques. The results were summarised as follows: (1) The IgG conc. in blood serum of epileptic patients increased as compared to the non-epileptic persons. (2) The IgG conc. in blood serum of non-epileptic and epileptic patients, studied by UV spectroscopy, showed similar results. The increase in IgG conc. in blood serum of epileptic patients is due to increased level of neuron-specific antibodies in the sera. (3) The molecular characterisation of IgG was studied by disc gel electrophoresis. A single protein band was observed in a normal person's pattern; whereas, the protein profile of IgG in abnormal serum samples (E1, E2 and E3) shows one or two protein bands of both IgG and IgA. Top Keywords Separation, Isolation, Purification and Characterisation of Protein IgG, Blood serum, Non-Epileptic and Epileptic Patients. Top |
Introduction Neurology deals with disorders of the nervous system and specifically, concerns with the diagnosis and treatment of all categories of diseases involving the central, peripheral and autonomic nervous systems, including their coverings, blood vessels, and all effector tissues, such as muscle. Neurological disorders are disorders that can affect the central nervous system (brain and spinal cord), the peripheral nervous system or the autonomic nervous system. |
Epilepsy is derived from the Greek word epilepsia, which in turn comprises epi (upon) and lepsis (to take hold of Seizure). Epilepsy is a common chronic neurological disorder that is characterised by recurrent unprovoked seizures. The latter are transient signs and or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. Epilepsy is usually controlled but not cured with medication, although surgery may be considered in difficult cases, not all epilepsy syndromes are lifelong and hence some forms are confined to particular stages of childhood. Epilepsy is not a single disorder, but rather a group of syndromes with vastly divergent symptoms, but all involving episodic electrical activity in the brain. It is classified in 5 types:
By their first cause (or etiology); By the observation manifestation of seizures, known as semilogy; By the location in the brain where the seizures originate; As a part of discrete, identifiable medical syndromes and by the event that triggers the seizures, As in primary reading epilepsy.
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Antibodies, also known as immunoglobulin are gamma globulin proteins that are found in the blood or other body fluids of vertebrates, and are used by the immune system to identify and neutralise foreign objects, such as bacteria and viruses. In other words, antibodies are glycoproteins. |
The latter are proteins that contain oligosaccharide chains (glycans) covalently attached to their polypeptide side-chains. Antibodies occur in two forms, a soluble form secreted into the blood and tissue fluids, and a membrane-bound form attached to the surface of a B cell that is called the B-cell receptor (BCR). The BCR allows a B cell to detect when a specific antigen is present in the body and triggers B-cell activation. Activated B cells differentiate into either antibody generating factories called plasma cells that secrete soluble antibody, or into memory cells that survive in the body for years afterwards to allow the immune system to remember an antigen and respond faster upon future exposures. Antibodies are, therefore, an essential component of the adaptive immune system that learns, adapts and remembers responses to invading pathogens. Antibodies are produced by a kind of white blood cell called B cell. Immunoglobulin G (IgG) is a tetrameric immunoglobulin, built of two heavy chains and two light chains. Each IgG has two antigen-binding sites. It is the most abundant immunoglobulin and is approximately equally distributed in blood and in tissue liquids, constituting 80% of serum immunoglobulins in humans. IgG antibodies are large molecules of about 150 kDa composed of four peptide chains. It contains two identical heavy chains of about 50 kDa and two identical light chains of about 25 kDa. |
The two heavy chains are linked to each other and to a light chain each by disulphide bonds. The resulting tetramer has two identical halves, which together form the Y-like shape. Each end of it contains an identical antigen-binding site. |
Top Materials and Methods Sample collection The blood sample of epileptic patients and normal (disease free) persons were collected from Ranbaxy Pathology Lab, Muzaffarnagar. Method of Sampling The serum samples of diseased and normal persons were incubated at 37–40°C for 30 min. The blood cells settled down in the vials and the serum was separated as a clear pale yellow liquid. The serum was isolated with the help of a pipette into fresh vials and the serum samples were stored at 4°C. Purification of IgG By Acrylic Acid Caprylic acid was used to purify mammalian IgG from serum, and hybridoma culture was supernatant by precipitation of non-IgG protein. Reagents 0.6 M sodium acetate- (pH 4.6) Caprylic acid (free acid)
Method In order to purify IgG, the serum (disease and normal) was centrifuged at 10,000 rpm for 20–30 min. The pellets were discarded and twice the volume of 0.06 M sodium acetate (pH 4.6) was added. Caprylic acid was added dropwise while stirring at room temperature. To each 25 ml of serum, 1.52 ml of caprylic acid was added and centrifuged at 4000 rpm for 20–30 min. The supernatant was retained and pellets were discarded. The supernatant was dissolved in phosphate buffer saline (PBS). By Ammonium Sulphate Precipitations Purification by caprylic acid method used prior to ammonium sulphate precipitation to yield a product of higher purity. Reagents: (Used ammonium sulphate precipitation) Saturated ammonium sulphate solution (pH 7.2). Normal saline: NaCI dissolved in double-distilled water. 10×PBS (phosphate buffer saline) pH 7.2 (100 ml).
Membrane changing solution: 0.4% NaHCO3 and 0.05 g EDTA in 100 ml double-distilled water. 1% BaCI2
Precipitation of globulin from serum Serum was diluted with saline and added saturated ammonium sulphate solution to final concentration of 45% (v/v). Required concentration of ammonium sulphate was calculated by using the formula: Concentration of ammonium sulphate = 100 (Sf-Si) 1-Sf Sf = final saturation Si = initial saturation The whole content was stirred at 4°C for 30 min and the precipitates were centrifuged at 3000 rpm for 130 min at 4°C. The precipitates were washed with 45% saturated ammonium sulphate solution and centrifuged again. The precipitates were redissolved in the same volume of PBS as the original serum. The solution was centrifuged to remove any insoluble material; the globulin was reprecipitated by using a final concentration of 40% saturated ammonium sulphate solution. The pellets were redissolved in minimum volume of PBS. The globulin isolated from serum was dialysed against four changes of PBS at 4°C. Activation of dialysis tubing Dialysis tubing of a suitable diameter and length according to sample volume was selected and submerged in the solution of 2% NaHCO3 + 0.05% EDTA and boiled for 10 min. The solution was discarded and boiled again in double-distilled water for 10 min. Activated dialysis tubing could be stored at 4°C for up to 3 months. Desalting of protein sample by dialysis The dialysis tubing was sealed from the end by a clip. The sample was filled using a micropipette and sealed at the other end, it was suspended with the help of a thread in a beaker containing 100 vol. of PBS. The beaker was kept on a magnetic stirrer and left at 4°C at least for 2 h. Like this at least four changes were given. To check whether dialysis is complete or not 1% BaCl2 solution was added into PBS. Appearance of precipitates showed that dialysis has been completed.
Sepharose-2B (gel permeation chromatography) Sepharose-2B (5 g dry beads) were suspended in 0.1 M Tris buffer (pH 7.0) and allowed to swell for 3–4 hrs at room temperature. Decant the excess buffer along with any suspended fine particles to obtain slurry of reasonable thickness. The column was fixed upright on a burette stand with the help of clamps. The outlet of the column was kept closed, a plug of glass wool was placed at the base of the column and a small volume of the buffer or water was poured. The slurry was poured gradually into the inner surface of its wall and was gently tapped to expel the air bubbles. The chromatographic media was allowed to settle down evenly and then the outlet was opened to drain out excess liquid from the column. The mobile phase was at the top of the peaking, and was drained out till the bed surface got exposed. The outlet was closed and the serum samples of cervical spondylitic patients and control was loaded over the bed surface with a pipette and the loaded sample was then allowed to enter the column by opening the outlet. A sufficient amount of buffer was added on top of the column and connected to the buffer reservoir. The fractions were collected manually and the protein content was obtained by monitoring absorbance at 280 nm. The graph of conc. of protein vs fraction number or elution was plotted. Estimation of protein concentration by UV spectroscopy Protein was determined by the method of UV spectroscopy at 280 nm using BSA (100–1000/l g) as a standard. Assays for Protein Estimation IgG antibody of a normal person and an epilepsy patient was taken in vials. The volume was made up to 3 ml in each vial with PBS. Absorbance was measured at 280 nm. IgG content was measured referring to a standard plot of BSA (100–1000 ug). Estimation of Protein by SDS-PAGE and Disc Electrophoresis The term electrophoresis describes the migration of charged particles under the influence of an electric field. Many important biological molecules, such as amino acid, peptide, protein, nucleic acid and nucleotide possess ion sable groups, therefore at any given pH, they exist in the solution as electrically charged species either as cation (+) or anion (−). Under the influence of an electric field these charged particles will migrate either to cathode or to the anode, depending on the nature of their net charge. When a potential difference (voltage) is applied across the electrodes, it generates a potential gradient E, which is the applied voltage, divided by the distance between the electrodes’ force is applied or molecule bearing a charge q coulomb is Eq. There is also a frictional resistance, which related the movement of the charged molecule, due to its, size and shape. The velocity v, of a charged molecule in an electric field is given by V=Eq/f All components were mixed and stored in a freezer. All components were mixed and the volume was 1000 ml. Method Resolving gel (8%) and stacking gel (5%) were prepared by adding the component described in the composition of gels. The apparatus was set; the resolving gel was poured in gel tubes and allowed to set. Now the stacking gel was poured and gel tubes were placed vertically on assembly, and allowed to polymerise. Sample buffer, in the amount, according to protein content was added to each sample. The protein was heated at 100°C for 5 min, and loaded in the respective wells. Electrophoresis was carried out at 0.02 amp in the presence of electrophoresis buffer. When the sample buffer reached at the bottom, the unit was switched off. The gel was immersed in fixing the solution for l-L hours. Now, the gel was immersed in a staining solution overnight and detained the next day by using the destaining solution. Top Results and Discussion Standard curve for estimation of protein concentration by UV Spectroscopy (at 280 nm) The protein standard curve was prepared by the method of UV Spectroscopy (at 280 nm). It has been seen that the standard curve is linear at 50–500 ug of BSA. At a certain point it falls below and above the standard line, although these points do not affect the results. The linear range of protein concentration was obtained between 50 and 500 ug (Figure 1). Comparative study of protein concentration in blood serum of a normal person and patient suffering from epilepsy The IgG concentration was calculated in blood serum of a patient suffering from epilepsy (mg/ml) by using the standard curve. The result of UV Spectroscopy technique (at 280 nm) showed the increased IgG concentration in a patient suffering from epilepsy, in comparison to the blood serum of a normal person. This may be due to an increased level of neuron-specific antibodies in the sera of the patient. Biophysical analysis of IgG in blood serum of a normal person and patients suffering from epilepsy by UV Spectroscopy Absorption spectra of IgG in the UV region of a normal person and a patient suffering from epilepsy were taken at 280 nm. The normal person showed the highest peak of absorbance at 0.300 nm and protein concentration at 2.5 mg/ml; while that of a patient suffering is shown in the following order (Figure 2): SDS-PAGE profile of a normal person and a patient suffering from epilepsy SDS-PAGE was carried out using 6–8% polyacrylamide gel. The protein bands were stained with Commassie Brilliant Blue (CBB) R250 staining solution overnight at room temperature. Disc I shows the protein band profile of purified IgG in normal serum sample. A single band (approx. 150 KDa) was observed in this pattern; whereas, the protein band profile of IgG in abnormal serum samples (E1, E2 an E3) shows one or two protein bands of both IgG and IgA (IgG 150 KDa, IgA 150–600 KDa). The standard molecular marker was applied for the determination of molecular weight of unknown protein bands. The range of protein markers are: Top Figures | Figure 1:: Standard curve of BSA
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| | Figure 2:: Comparative study of protein in a serum sample of a non-cervical spondylitic (NCS) and that of patients suffering from cervical spondylitis- (CS1), (CS2) and (CS3)
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Tables :
| | Composition of Resolving Gel 8% | 12 ml | | Distilled water | 5.0 ml | | 30% Acrylamide | 4.0 ml | | 1.5 M Tris (pH-8.8) | 3.15 ml | | 10% SDS | 0.125 ml | | 10% APS | 0.125 ml | | TEMED | 0.005 ul | | Stacking Gel 5% 4 ml | | | Distilled water | 2.8 ml | | 30% Acrylamide | 0.625 ml | | 1.5 M Tris (pH-6.8) | 0.5 ml | | 10% SDS | 0.04 ml | | 10% APS | 0.04 ml | | Acrylamide Mix | | | 29.2% Acrylamide + 0.8% bisacrylamide | | | Agar Solution | | | 500 mg/25 ml of distilled water. | | | Sample Buffer | | | Tris HCL buffer (pH-6.8) | 2 ml | | 10% SDS | 3 ml | | Glycerol | 0.2 ml | | Beta-Mercapto ethanol | 0.2 ml | | Bromophenol blue | 1 ml |
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| | Electrophoresis Buffer | | | Tris | 3.0 g | | Glycine | 9.8 g | | SDS | 0.7 g |
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| | Staining Solution | | | Coomassie brilliant blue | | | Coomassie blue | l.4 g | | Methanol | 200 ml | | Glacial acetic acid | 25 ml | | Distilled water | 265 ml | | Volume was | 500 ml | | Distaining Solution | | | Methanol | l50 ml | | Acetic acid | 75 ml | | Distilled water | 875 ml | | Volume was made to | 1000 ml | | Fixing Solution | | | Methanol | 30 ml | | Acetic acid | 10 ml | | Distilled water | 60 ml | | Volume was made to 100 ml. | |
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| | Patients suffering from Epilepsy | Peak absorbance (nm) | Protein concentration (mg/ml) | | E1 | 0.374 | 2.60 | | E2 | 0.619 | 4.20 | | E3 | 0.906 | 6.20 |
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| | Myosin, (Rabbit muscle) | - 205,000 Da | | Mammalian IgG | - 150,000 Da | | Phosphorylase b | - 97,400 Da | | BSA | - 66,000 Da | | Insulin | - 3,500 Da |
| | | Table 1:: Standard curve of BSA (2 mg/ml) for estimation of protein concentration by UV Spectroscopy at 280 nm
| | S. No. | BSA vol (ul) | BSA Conc. (ug) | Distilled Water (ml) | O.D at 280 (nm) | | 0 | 00 | 00 | 3.000 | 0.000 | | 1 | 25 | 50 | 2.975 | 0.007 | | 2 | 50 | 100 | 2.950 | 0.014 | | 3 | 75 | 150 | 2.925 | 0.022 | | 4 | 100 | 200 | 2.900 | 0.028 | | 5 | 125 | 250 | 2.875 | 0.037 | | 6 | 150 | 300 | 2.850 | 0.053 | | 7 | 175 | 350 | 2.825 | 0.062 | | 8 | 200 | 400 | 2.800 | 0.059 | | 9 | 225 | 450 | 2.775 | 0.063 | | 10 | 250 | 500 | 2.750 | 0.072 |
| | | Table 2:: Comparative study of protein in serum sample of non-epileptic (NE) and that of patients suffering from epilepsy-(E1), (E2) and (E3)
| | Fractions | O.D(NE) | O.D(E1) | O.D(E2) | O.D(E3) | | 1 | 0.054 | 0.012 | 0.007 | 0.089 | | 2 | 0.079 | 0.127 | 0.239 | 0.114 | | 3 | 0.236 | 0.346 | 0.393 | 0.297 | | 4 | 0.246 | 0.368 | 0.414 | 0.425 | | 5 | 0.300 | 0.374 | 0.383 | 0.506 | | 6 | 0.285 | 0.147 | 0.159 | 0.564 | | 7 | 0.275 | 0.139 | 0.061 | 0.357 | | 8 | 0.255 | 0.130 | 0.030 | 0.008 | | 9 | 0.184 | 0.097 | 0.013 | 0.024 | | 10 | 0.173 | 0.071 | 0.008 | 0.022 | | 11 | 0.265 | 0.060 | 0.008 | 0.031 | | 12 | 0.125 | 0.043 | 0.005 | 0.037 | | 13 | 0.150 | 0.027 | 0.001 | 0.034 | | 14 | 0.268 | 0.023 | 0.002 | 0.035 | | 15 | 0.204 | 0.035 | 0.008 | 0.077 | | 16 | 0.134 | 0.024 | 0.002 | 0.020 | | 17 | 0.115 | 0.012 | 0.000 | 0.016 | | 18 | 0.104 | 0.013 | 0.000 | 0.027 | | 19 | 0.105 | 0.021 | 0.001 | 0.031 | | 20 | 0.100 | 0.038 | 0.006 | 0.092 | | 21 | 0.095 | 0.010 | 0.004 | 0.037 | | 22 | 0.087 | 0.047 | 0.001 | 0.036 | | 23 | 0.039 | 0.029 | 0.003 | 0.039 | | 24 | 0.034 | 0.033 | 0.002 | 0.034 | | 25 | 0.024 | 0.025 | 0.001 | 0.024 | | 26 | 0.027 | 0.018 | 0.001 | 0.027 |
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