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Experiment-6 : Autoantibody profiling in diseased samples
Experiment 6: Autoantibody detection in diseased samples
Objective: To detect the autoantibodies in tumor tissue samples using serological proteome analysis
Autoantibodies are defined as an immunoglobulin produced by an immune system in response to antigenic constituent of one’s own tissue. Autoantibodies may also be generated when immune system is not able to distinguish between self and non-self proteins. There are a number of mechanisms that can trigger the production of antibodies; prolonged exposure to toxic chemicals, the disturbance caused in immuneregulation for antigens might allow the production of autoantibodies. Some of the autoantibodies may act as useful biomarkers and indicators of disease activity providing prognostic information.
Current proteomic technologies aids in identification and characterization of individual proteins and their potential use as biomarkers. Proteomic approach can be implemented for the identification of autoantibodies to the tumor antigen for humoral response. Protein microarray is a ubiquitious method which involves an arraying device, and depends on the affinity tags as antibodies, a detecting device and bioinformatic analysis. A hybrid method which is a fusion of gel electrophoresis and western blotting known as Serological proteome analysis (SERPA) is much used for the identification and characterization of proteins. The Serological proteome analysis is a promising method that helps in identification of the autoantibodies in cancer patients. The extracted protein samples from tissue are separated through 2-D gel-electrophoresis; these resolved protein spots are electro-transferred to the nitro-cellulose membrane. Nitrocellulose membrane is then incubated with the serum of healthy patients. The membrane is incubated with Anti IgG immunoglobulin conjugated with horseradish peroxidase followed with the ECL reaction and autoradiography for immunodetection. The proteins are identified by excision of spots from preparative stained gel for mass spectrometric analysis through MALDI TOF /TOF.
 Antibodies are produced by immune system to attack external antigens where as autoantibodies are produced in response to antigens on the surface tumour cells. Autoantibodies that are associated with human diseases depict the hallmark of a typical immune response and aid to diagnose autoimmune diseases. Hence we need to test a number of autoantibodies to ascertain diagnosis. Individual diagnosis is important for various diagnosis and therapeutic decisions. Auto-antibodies against one or more of the tumour-associated antigens can play an important role in indicating the stage of the cancers. A proteomic approach is employed for the identification of autoantibodies as tumour biomarkers eliciting humoral response; for instance microarray approach is used to analyse blood samples for the presence of specific autoantibodies which are directed against glycoproteins [Wandall et al. (2009) Cancer Res: 70(4):1306-13]. These glycoproteins provoke immune response by undergoing abnormal glycosylation.However we are employing a fusion of common and a very basic two dimensional electrophoresis and western blotting technique for the identification of such autoantibodies
Gel electrophoresis separates protein and these separated proteins are transferred on the nitrocellulose membrane, which is incubated with the serum of the patients. The antibodies present in the serum will act as affinity substrate and binds with the protein present. Adding an anti - immunoglobulin conjugated with HRP antibodies and examining through ECL reaction will help in immunodetection. Protein identification and characterization is done using mass spectrometry.
       I.          Collection of tissue and serum samples
     II.          Preparation of protein lysate
   III.          Western Blotting
 IV.          2-D gel electrophoresis
    V.          Serological proteomic analysis (SERPA)
 VI.          Protein Identification and Characterization
I.  Collection of tissue and serum samples :
Serum and Tissue samples of cancer patients are collected after the informed consent is obtained. These samples are stored in -80˚C after excision. Samples from tumors at different stages of growth are also collected to investigate the progression of cancer in that tissue. After surgical resection, these samples are snap frozen using liquid nitrogen and immediately stored at -80oC.
II. Protein lysate preparation  

To prepare samples for western blotting and serum proteomic analysis , the serum and tissue sample are lysed properly using a homogenizer or a sonicator to release protein.This helps protein to migrate properly through the separating gel.

III.Western blotting
Western blotting also known as ‘immunoblotting’ is a powerful analytical technique, widely used in biological research for the detection of specific proteins.The blotting refers to the transfer of protein which is separated by gel electrophoresis depending on their molecular weight onto the surface of membrane (PVDF or nitrocellulose membrane). In this technique an antibody is used to specifically detect its antigen. The macromolecules are separated using gel electrophoresis and then resolved proteins are electrotransferred onto the matrix, which is membrane. The membrane blocking is achieved by incubating the membrane with albumin or milk proteins to prevent any non-specific binding of antibodies. The transferred protein is them complexed with a primary or a secondary antibody which is labeled with an enzyme probe. Addition of a relevant substrate to the enzyme produces a detectable compound on the membrane. The most sensitive method of detection apart from visual or colorimetric detection is using a chemiluminescent substrate which reacts with the enzyme to produce light as a by product. This light output can be captured using film, CCD camera or a phosphoimager that is designed for chemiluminescent detection. Fluorescence imaging system is another method of detection which uses fluorescently tagged antibodies. All these detection methods are designed such that they correlate with the abundance of the antigen on the membrane. The results obtained are easy to interpret and helps in identification of proteins.
Western Blotting provides qualitative as well as quantitative information about a protein. This technique aids in studying the proteomic profile of proteins. Proteins when bind with the membrane they are more accessible for immunological or biochemical analyses, quantitative staining and western blotting is hence powerful technique for the visualization and identification of proteins. The proteins are separated depending on their molecular weight and are detected by the specific antibody confirming the identity of a target protein.
 Fig.1: Schematic representation of the Western blotting procedure.
IV. Two dimensional gel-electrophoresis
Electrophoresis is method whereby charged molecules specially proteins and nucleic acids migrate in response to an electrical field. Two dimensional electrophoresis separates protein in two different steps, based on two different principles ; isoelectric focusing in which is proteins are separated on the basis of their isoelectric point and the second dimension SDS PAGE , where protein are separated according to their molecular weight.     The second dimension is done on SDS PAGE i.e. sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE). Under applied electric field the proteins are separated on the gel strip followed by second dimension after equilibration, where proteins are separated on the basis of their molecular weight.  For better protein separation and to avoid protein aggreagation Tris-glycine system at high pH is used as a running buffer. Two dimensional electrophoresis is discussed in detial in previou experiments.
Fig. 2: SDS-PAGE: Proteins separated on the IPG strips on the basis of their charge are coated with SDS to give them a uniform negative charge. In the presence of an electric field, these proteins then get separated only on the basis of their molecular weights.
V. Serological Proteome Analysis ( SERPA)
SERPA is a hybrid method which includes two dimensional electrophoresis along with the affinity reagents for detection of autoantibodies. Proteins resolved on the gel are electro-transferred to nitrocellulose membrane, and then incubated with serum from healthy individuals (1:300 dilutions). Immunodetection can be done with antihuman IgG antibodies conjugated with horseradish peroxidise followed by ECL reaction and autoradiography. Films obtained are scanned with a densitometer and used for image analysis.
VI. Protein Identification and Characterization
Protein spots were excised from preparative gels which are loaded with protein obtained from the tumor tissues. In-gel digestion is performed for the relevant protein spots which correspond to antibody reactivity and are specific to healthy in serum (visualized by staining of SDS-PAGE).
Mass spectrometric method is used for the characterization of proteins detected. Mass spectrometric analysis is done using MALDI TOF /TOF. One µl of sample prepared in suitable matrix in 1:1 ratio is spotted on the MALDI plate and is subjected to fragmentation for proteomic profiling through MS/MS. The fragmentation pattern obtained is blasted in MASCOT for determination of the sequence in SwissProt database.


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