Molecular Biology: Fluorescence Activated Cell Sorting - Essay Example

Molecular Biology Monoclonal antibodies are used in research, diagnostics and therapeutics. Monoclonal antibodies are issued from one single cloneof cells. Monoclonal antibodies from mouse origin are easy to produce but their therapeutic availability is restricted by their antigenicity. Human monoclonal antibodies are produced by humanising mouse monoclonal antibodies. Monoclonal antibodies can be obtained by intraperitoneal injection into mice and collection of ascetic fluid produced. Since it is difficult to to remove antigenicity from the mouse, in vitro immunisation (IVI) protocol of human peripheral blood mononuclear (PBMC) cells for generating human antigen specific antibodies was developed. For monoclonal antibody, mice are immunised with purified protein. Mouse splenocytes are collected after the immunisation process and fused with myeloma cells. To obtain monoclonal antibodies individual B cells are fused to myeloma cells and isolated by serial dilution resulting to a fusion product or hybridoma cell line of which can produce one specific antibody for extended periods of time by tissue culture. These hybridomas can be screened and best clones cultured in standard tissue culture facilities. The hybridoma cell line obtained for a given antibody was injected into the peritoneal cavity of mice where it grew and simultaneously produces the antibodies. Ascetic fluid containing the antibodies was harvested from the peritoneal cavity after a period of time. One fusion may produce 1000hybridomas therefore one must choose the most appropriate. This might be the highest infinity mAb but could be the most stable, the least cross reacting, the highest specificity depending on what is required. The advantage of using monoclonal antibodies is that they are monospecific, they tend to reduce cross reactivity and useful in diagnostics such as tissue typing. They are highly reproducible, can use relatively impure antigens to immunise animas, theoretically have limitless supply and can manufacture using recombinant DNA technology plus phage display libraries to produce fully human antibodies of any specificity. Labelling antibodies is useful as it enables detection of antibodies. Currently, the most commonly used are fluorescent labels, enzyme labels, chemiluminescent labels and radioactive labels. These are sometimes amplified using Avidin-Biotin Conjugate system. Some common fluorescent labels include, Fluorescein isothiocyanate, DAPI, Phycoerithrin and Texas red. When using enzyme labels, an enzyme is covalently attached to the antibody and converts a colourless synthetic substrate to a coloured product. The most common are alkaline phosphatase, horseradish peroxidase (HRP) and urease. When using HRP is efficient. The carbohydrate on the HRP is linked to the amino groups on the protein via Schiff base formation. HRP has a high percentage of carbohydrate residues which are not too intimately linked to the active site. Controls should be standard that is positive and negative tissue controls. The major use is detecting tumour antigens in tissues, detecting auto-antibodies in autoimmune states and detecting virus particles in tissue sections. 2. Fluorescence activated cell sorting (FACS) uses the basic principle of rapid analysis and sorting of individual cells, cell by cell, in a flowing stream according to cell size and granularity and cell fluorescence. Measurement of cell size and granularity is sufficient to differentiate major categories of leukocytes in peripheral blood. Adding fluorescent probes to the cells enables quantitation of specific structures. The cell suspension is labelled with monoclonal antibodies to the surface of the antigen. The fluorescence is created by an appropriate dye attached to a monoclonal antibody (Coico & Sunshine, 2009). When compared to microscopes FACS is fast as it uses less than a minute per sample as opposed to 5-10 minutes for the slides, can achieve over 106 cells per determination as opposed to 100 per slide, can analyse multiple parameters per determination, has high objectivity and reproducibility and offers automated quantification set of parameters. FACS is used in leukaemia diagnosis using CD10 markers for Acute myeloid leukaemia (AML) (Coico & Sunshine, 2009). An analytical flow cytometer take in a suspension of monodisperse cells and run them one at a time past a laser beam. When the laser strikes the cell, particulate light scatter happens. Forward Angle Light Scatter (FALS) relates to the size of the whole cell. The Right Angle Light Scatter (RALS) relates to the granularity of the cells. The cells are quantitated as each cell passes through the laser beam and scattered. Limitation to sensitivity is caused by naturally fluorescent components of cells that set a background fluorescence intensity. After information is obtained, the sample is discarded (Coico & Sunshine, 2009). Plotting Forward Scatter against Side Scatter on a linear scale will give information about the morphology about the human blood leukocytes. Gating is used to identify populations for instance in analysing lymphocytes in a sample of blood, a gate is placed on the lymphocytes to show only data within the gate. The gate can either be a bitmap or rectilinear gate (Coico & Sunshine, 2009). A fluorochrome is a fluorescent marker. It is a molecule that fluoresces when a laser light hits it. Two approaches can be used. First, a fluorescent label attached to a mAb which will bind to the cell marker of interest and can be used to identify a particular cell type based on the individual antigenic markers of the cell and can be done by adding the conjugated Abs to a mixture of cells. The mAb approach can be used to label any cell marker, can be dual label with two or more fluors of different colours e.g. CD4, CD8, CD45, CD10, HLA-DR, cell cycle and apoptosis markers. Secondly a fluorescent label with a specific affinity or property in its own right (Coico & Sunshine, 2009). The nature of leukaemia can be understood can be understood by identifying specific proteins on their surface using antibodies. Acute lymphoblastic leukaemia in children usually CD10 (CALLA) positive although would tend to also look at CD19, CD5 and HLA. Adult T cell leukaemia is usually CD4 and CD25 positive and others such as the myelogenous leukaemia, hairy cell leukaemia. Malignant astrocytoma cells are antigenically distinct from normal tissue to be recognised tumour specific T cells. The tumour antigen is any protein produced in a tumour cell that has an abnormal structure. Tumour antigens can be tumour specific or tumour associated antigens. Tumour specific antigens are caused by mutation of proto-oncogenes and tumour suppressors leading to abnormal protein production. Tumour associated antigens are a result of mutation of other genes which are not related to tumour formation. They are often expressed by tumours from different individuals unlike tumour specific antigens are encoded by point mutations and more patient specific. Tumour specific antigen is predicted to contain HLA binding epitopes (Coico & Sunshine, 2009) 3. Antibodies have great diversity of antigen combining sites is due to the highly variable amino-terminal protein sequences of both light and heavy chain polypeptides. The antibodies have variable region gene (V gene) and constant region gene (C gene). At the DNA, RNA or protein level, during differentiation of an individual antibody forming cell, one of a number of V genes would be associated with the C gene. During rearrangement of the genes when a cell commits to become a B cell making it a light chain, one of the V genes is brought next to the J regions. This is a recombination event that removes an intron between the V and J regions (Chaudhuri, 2013). In heavy chain gene, there are many C genes. Each C gene is composed of axons, V region genes, J exons and D exons. There are two rearrangements at the DNA level. One of the D regions is brought next to the rearranged DJ region. This occurs by removal of introns between V, D and J regions (Chaudhuri, 2013). MAbs are isolated by phage display, the gene is isolated directly and variable region genes can be retrieved from hybridoma cells. Libraries of genomic clones constructed in a phage vector were size fractionated and screened with gene fragments or synthetic oligonucleotide probes to identify variable regions heavy and light chain genes. This approach has the advantage that the Ig promoter and Ig enhancer can be recovered as a single fragment using suitable restriction sites which are useful in subsequent expression studies. Genes can also be recovered from mRNA through cDNA cloning. In this case there is abundant mRNA therefore only small libraries of cDNA are required. The libraries are also screened with gene fragments or oligonucleotides to isolate the required genes. PCR allows rapid isolation of variable region genes through PCR amplification. However, PCR would cause introduction of errors into sequence due to replication errors or cross examination but can be overcome by sequencing multiple independent clones (Chaudhuri, 2013). Human anti-mouse antibody response (HAMA) is an immune response encountered when administering murine MAbs to humans. HAMA prevents repeat administration of antibody due to formation of immune complexes which are rapidly cleared form the body rendering the repeat therapy ineffective. HAMA responses may result in adverse reactions such as allergic reactions. Production of chimeric or fully humanised antibodies is used to solve this problem (Chaudhuri, 2013). In making antibodies more human are by replacing constant domains of the antibody with constant domains of the antibody. The resulting chimeric antibody contains only the variable regions of murine origin and would therefore be expected to be less immunogenic in people. Many chimeric antibodies retain the full antigen binding ability of the parent murine antibody as well as taking on the constant region effector functions of the human antibody used (Chaudhuri, 2013). Chimeric genes are created by by exchanging the variable regions of the human antibody heavy and light chains genes. The antigen specificity is identical to parental mouse antibodies but the in vivo half life and effector functions are human. Chimeric antibodies are used to treat cancer and transplant rejection. When mouse CDRs are engrafted onto human framework region and thereby recapitulate the antigen binding characteristics of the original mouse antibody resulting to a >90% human humanised antibody. Afutuzumab is a humanised monoclonal antibody that targets CD20 and kills B cells. It is used for treatment of chronic lymphocytic leukaemia. Rituximab also known as chimeric is a humanised anti-CD20 MAb. CD 20 is found on B-lymphocytes and especially follicular non-hodgkins lymphoma. It kills via ADCC and complement as well as apoptosis induction. Rituximab can be used in similar CD20 expressing tumours such as chronic lymphocytic leukaemia and small cell leukaemia. Side effects include fever, chills and nausea (Chaudhuri, 2013). 4. Transcription is the first step in gene expression in transcription into RNA. Transcription is initiated with the binding of enzyme RNA polymerase to a specific DNA sequence at the beginning of the coding region referred to as promoter. Transcriptional factors control transcriptional regulation by binding to cis-regulatory sites. Protein modification such as acetylation, farnesylation, myristoylation, palmitoylation and phosphorylation assist in localizing the enzyme in various cell compartments. Transcription termination is also determined by specific DNA sequences located downstream of coding region (Sikdar, Todd & Bier, 1989). E.coli has a potential to become a recombinant source of virtually any protein of industrial interest. One major disadvantage regarding utilising genetically modified E. coli as a source of proteins is intracellular accumulation of recombinant protein as inclusion bodies. Another disadvantage is the inability of E.coli to perform post-translational modifications of recombinant eukaryotic proteins. E.coli do not have the ability to glycosylate, amidate or acetylate proteins. Eukaryotic proteins normally subjected to such post-translational modifications when produced in their natural source are devoid of modifications when produced in E.coli. Lack of post-translational modification may or may not have a significant effect upon biological activity of the heterologous protein. The normally glycosylated glycoproteins β-interferon and interleukin-2 when produced in E.coli, are biologically active despite being non-glycosylated. Absence of glycosylation may alter biological characteristics of the recombinant protein. Absence of a carbohydrate moiety can result in modification of biological activity of the molecule, alteration of circulatory half life and altered immunogenicity (Sikdar, Todd & Bier, 1989). Recombinant proteins produced in E.coli may contain extra amino-acid residue at their N-terminal end since translation is initiated by N-formylmethionine residue. The presence of an extra N-terminal methionine in such recombinant proteins may alter their biological characteristics. The bacterial plasmids used for cloning and a range of techniques can be used depending on the bacterium. Some bacteria need plant cell like approaches for example Pseudomonas spp. The major problem tends to reside with exopolysaccharide producing bacteria which can interfere with other procedures but can be removed by CTAB (Sikdar, Todd & Bier, 1989). Yeast cloning is easy as cloning bacteria. Mammalian cell cloning can grow in hollow fibre reactors and produce mg Ab per reactor. However, is slower and more expensive. When extracting mammalian DNA, it should be aim to avoid damage of DNA by endogenous enzymes. All tissue releases autolytic enzymes upon death therefore it is recommended to freeze as soon as possible. It is grinded in a pestle and mortar and free cells are centrifuged and washed. The membranes are disrupted with proteinase K and SDS. The next step is to suspend the buffer extract in equal volume phenol. Centrifuge and remove aqueous layer, ammonium acetate is added and cold ethanol to precipitate the DNA. The precipitate is then washed in ethanol (Sikdar, Todd & Bier, 1989). Bibliography Sikdar, S. K., Todd, P. W. & Bier W., 1989. Frontiers in Bioprocessing, Volume 1. CRC Press. Chaudhuri. K., 2013. Recombinant DNA Technology. TERI Press. Coico, R. & Sunshine, G., 2009. Immunology: A Short Course. John Wiley & Sons. Read More