Immunoassay Techniques: Fluorescence Resonance Energy Transfer and Western Blotting - Essay Example

Street Address Zip Email Phone A Comparison of Immunoassay Techniques: Fluorescence Resonance Energy Transfer and Western Blotting Student Name Immunoassays are biochemical tests that measure levels in a biological substance such as urine or serum utilizing the reaction of antibody to its antigen. Antibodies bind in a certain manner to their antigen, a quality utilized by the assay. The type of antibody most used is the monoclonal as it binds to one specific site on the antigen making it easier to distinguish among other molecules. A variety of methods can indicate the quantity of the antibody or antigen with the most common being the labeling of the antibody or antigen. This can be done with an enzyme (enzyme immunoassay EIA), radioisotopes such as I-125 Radioimmunoassay (RIA), or fluorescence. Another method is the Western Blot. Immunoassays are homogeneous or heterogeneous and can be competitive and noncompetitive. I "In the competitive immunoassay, the antigen in the unknown sample competes with labeled antigen to bind with antibodies. The amount of labeled antigen bound to the antibody site is then measured. In this method, the response will be inversely proportional to the concentration of antigen in the unknown. This is because the greater the response, the less antigen in the unknown was available to compete with the labeled antigen."(Wikepedia, Immunoassay) Noncompetitive immunoassays are called "sandwich assay." In this method the antigen in the unknown is bound to the antibody site and the labeled antibody is bound to the antigen. A measurement of the labeled antibody is taken. The results will be directly proportional to the concentration of antigen. In heterogeneous assays the unbound antibody must be removed with a reagent, but this step is not necessary with the homogeneous and thus is more convenient and quicker to use. The transfer of proteins to gel membranes is to provide easier access with probes of which the most common is the antibody. "The power of the technique lies in the simultaneous detection of a specific protein by means of is antigenicity and its molecular mass." (Rybicki 1996) For the enzyme-assisted immunoelectroblotting (IEB) or Western blot the following equipment is typical. A vertical slab gel apparatus Nitrocellulose paper and filter paper, blotting paper cut to size Blotting electrodes Blotting buffer -20% methanol and pH above 7.0 Nappy liners-to minimize surface area exposed to electrode The protein transfer is accomplished by dipping the polyacrylamide gels into the transfer buffer, which is then laid onto nitrocellulose paper that is pre-wetted placed on three layers of wet filter paper that rests on the Anode (+ve electrode). The gel is then overlaid with three wet filter papers and the Cathode (-ve electrode). It is imperative to make sure there are no bubbles between the layers. Next the completed package is placed on a plastic tray, anode side down. The Anode and Cathode are connected to a power pack with current of 500mA applied for approximately thirty minutes for transfer to occur. The nitrocellulose can be stained with Amido Black, Coomassie Brilliant Blue or Ponceau S staining. When using the IgG immunoglobulin proteins in Western Blotting electrophoretically separated standard proteins are used to estimate the size of the IgG protein. The IgG protein is multimetric with two heavy chains and two light chains. The heavy chains are indicated by 5 very dark markings arranged horizontally across next to the 50 kilodalton (kDa) standard protein, so the weight of the IgG protein is assumed to be 50 kilodaltons. Further down the nitrocellose membrane are five more markings much lighter near the 25 kilodalton area which are the light chains. The total of the two chains provides the molecular weight of the IgG protein, 150 kilodaltons. The protein IgG is one of the five major immunoglobulins or antibodies, which constitute the gamma globulin portion of blood proteins (Marieb, 1989 pg 687). IgG is a monomer structure and is of interest to scientists because it constitutes about 80% of the human antibodies. It also crosses the placenta and provides passive immunity to the fetus. To analyze Cytochrome C with the Western blot it is necessary to use the anti-Cytochrome C antibody as Western blotting is not suitable for the Cytochrome C antibody. Found in mitochondria, Cytochrome C is a 15 kDa protein functioning as an electron carrier in the cell respiration chain. It is found in a variety of species including animals, plants and unicellular organisms, which make it attractive for studying evolutionary divergence. Cytochrome C is a critical protein in the apoptotic pathway. Apoptosis is a controlled form of cell death used to kill cells in the process of development or in response to infection or DNA damage. (Wikipedia) This attribute focuses attention on Cyctochrome C in cancer research. The main disadvantages to Western Blotting are that it is time consuming, especially if it is necessary to run the test again due to inconclusive results. It can take up to an hour or more depending on the protein. The Western Blot is more expensive than other assays due to the materials needed, though some can be reused such as the nappy liner pads and membranes can be washed in saline solution. The advantage is that is can produce a great deal of protein for examination. Fluorescence resonance energy transfer (FRET) is an energy transfer mechanism between two fluorescent molecules (Wikipedia). It is a radiationless transfer that utilizes a fluorescent donor and an acceptor. The donor is stimulated to a specific fluorescent wavelength using two dipoles. The excited state is transferred to the second acceptor molecule and the donor returns to its normal state. The donor is the dye that absorbs the energy and the acceptor, a chromophore. Donor J() Acceptor Fluorescence Absorption Overlap Integral figure from(anatomy.usyd.edu). The overlap integral (J) represents the degree of overlap between the donor fluorescence spectrum and the acceptor absorption spectrum. Materials required for the FRET are a protein, which is labeled specifically and uniquely with a donor and acceptor probe, a spectrometer and a device to measure the fluorescent intensity. For FRET to occur clearly certain conditions must be met; The donor probe must have a high quantum yield The emission spectrum of the donor probe must overlap considerably the spectrum of the acceptor probe (overlap integral) There is an appropriate alignment of the absorption and emission moments and their separation vector (embodied in kappa square). The donor and acceptor must be within 10.5 x Ro from each other. (anatomy.usyd.edu). A series of equations is then used to figure donor quantum yield, FRET efficiency, overlap integrity, kappa square and Forster Distance. M.N. Kronick and P.D. Grossman described a "sandwich" assay technique and the fluorescent excitation transfer immunoassay using phycobiliprotein fluorescent dyes. The proteins are stable and hydrophilic, have very high absorbability with excitation and emission bands across the spectrum. They are linked to antibodies using conventional cross-linking reagents. (Kronick, Grossman, Clin. Chem, 1983) Phycoerythrins RPE) are light-harvesting phycobiliproteins that can be isolated from red, blue-green and crytomonad algae. RPEs are ideal for multiple labeling or for double labeling in flow cytometry using single excitation lines. FRET is a preferable method for testing IgG due to the two light chains being difficult to view in the Western Blot. RPEs allow for more distinct images and precise measurements. The advantages and disadvantages of using FRET are fairly equal. The advantages are, FRET is relatively cheap in comparison to other immunoassay techniques Very efficient in measuring changes in distances Distance between molecules is measured in a solution Only a few M of labeled proteins Measurement is rapid after labeling molecules Distance or changes can be measured in a complex of molecules. (FRET spectroscopy n.d.) Disadvantages, The orientation factor impairs precise measurement When measuring distance changes between two probes, the result is a scalar and gives no indications of which probe moves (donor or acceptor) The presence of free labels in solution could mask a change in energy transfer These measurements give the average distance between the two probes (FRET spectroscopy n.d.) The external illumination requirement of FRET needed to initiate fluorescence transfer can lead to background noise resulting from the direct excitation of the acceptor. This is known as photobleaching. A technique called Bioluminescence Resonance Energy Transfer (BRET) was developed to correct this consequence. The Western Blotting and FRET immunoassay techniques are both valuable tools for examining proteins. Depending on the purpose for the research either technique is accurate in respect to specific proteins with FRET producing cheaper and faster results. The Western Blot is excellent for quantitive needs. Works Cited Berney, C, Danuser, G. "FRET or NOFRET: A Quantitive Comparison." Biophysiology Journal (2003) Accessed April 19, 2006 < http://www.medscape.com/medline/abstract/12770904prt=true Marieb, Elaine N. Human Anatomy and Physiology. New York: The Benjamin Cummings Publishing Company, Inc. Rybicki, E.P. and Maud Purves. "Enzyme-Assisted Immunoelectroblotting (IEB or Western Blotting." Molecular Biology Techniques Manual. Third Edition (1996) Date Accessed April 16, 2006 Read More