Antibody Binding of Antigens

Antibodies are involved in the immune response. They're made up of two light polypeptide chains and two heavy polypeptide chains bonded together. Antibodies have a variable region which acts in a similar way to the active site of an enzyme. Each antibody has a variable region that is the complementary shape for a particular antigen and

Affinity chromatography involves the property of bio-recognition for the separation of proteins based on the reversible interaction between the protein and the specific ligand bound to a chromatography matrix. It enables us to purify bio-molecule on the basis of its biological function and its chemical structure. The goal of the experiment was to gain hands-on experience in protein purification by using AKTA FPLC system. The IgG antibody contained in the mouse antiserum was purified by protein G affinity chromatography using a HiTrap protein G HP column. The chromatogram was then obtained and analyzed to ensure the separation of IgG antibody. Such purification of the protein before sample preparation is usually done to avoid any undesired interactions.

The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).

In this experiment, the primary antibodies against DAT were generated from the species Rattus norvegicus (the lab rat). The secondary antibodies (Chicken Anti-RatDAT) synthesized against Rat Anti-DAT, were generated from Gallus gallus domesticus (chickens). The Chicken Anti-RatDAT antibodies were synthesized by injecting certain purified rat antibodies into a chicken. As a result, a generation of the monoclonal antibodies Rat Anti-DAT, was essential to detect the DAT segment of the protein. Likewise, the polyclonal antibody Chicken Anti-RatDAT, linkage to the Fc portion of Rat Anti-DAT. Secondary antibodies are known to assist in the sorting, detection, and/or purification of target antigens by attaching to a primary antibody, that directly attaches to the target antigen.

Second, in order to further confirm the information about characteristics and function of the targeting protein that we have

The predicted antigenic regions were chimerically expressed in a bacterial system. Briefly, two sets of PCR primers were designed according to gD gene sequence available in GenBank (accession number: NC_001847) to amplify gene fragments encoding aa 20-160 (nucleotide 118953～119375, Δ gD1) and aa 257-344 (nucleotide 119664～119927, Δ gD2), respectively. The primers sequences for amplification of Δ gD1(P1 and P2) and Δ gD2 (P3 and P4) were listed in Table. 1.

Figure 1 Gel Electrophoresis for Replication Taster PTC. The gel is composed of an ethidium bromide stained 3% agarose gel demonstrating DNA fragments which were a depiction of PCR amplification. The agarose gel contains nine loading samples, including from left to right, the MW marker lane 1 precision mol mass standard, lane 2 TB undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 3 TB digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 4 TB A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 5 TB A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye), lane 6 MG undigested PTC (5µl of DNA, 5µl of master mix P, and 2.5µl of loading dye), lane 7 MG digested PTC (5µl of DNA, 5µl of master mix P, 2µl Fnu4HI, and 3µl of loading dye), lane 8 MG A(L)DH G (10µl DNA, 10µl master mix G, and 5µl loading dye), lane 9 MG A(L)DH A (10µl DNA, 10µl master mix A, and 5µl loading dye).

Second, for the western blotting, the primary antibodies that used to recognize the protein of interest (FLAG-PTEN) are rabbit anti-PTEN antibodies and the secondary antibodies that bind to the primary antibodies and convalently linked substrate to produce visible signal are goat anti-rabbit conjugated HRP (horseradish peroxidase) antibodies; for immunoprecipitation, the antibodies used to immobilize protein of interest are anti-FLAG monoclonal antibodies from morine cell. As introduction above, those antibodies come from different species. In order to be successful in western blotting, the primary antibodies recognize the protein of interest as well as bind to it. Consequently, rabbit anti-PTEN antibodies were used to bind to FLAG-PTEN. Meanwhile, goat anti-rabbit conjugated HRP antibodies, the secondary antibodies,

They only target part of the pathogen to provoke an immune response. This may be done by isolating a specific protein from a pathogen and presenting it as an antigen on its own. These vaccines can be created by genetic engineering as well, via a gene coding. A gene coding for a vaccine protein is inserted into another virus, when the carrier virus reproduces or when the producer cell metabolizes the vaccine protein is created as a product. The end result is a recombinant vaccine. Conjugate vaccines are made using pieces from the coats of bacteria. They are a combination of two different components. The coats are chemically linked to a carrier protein and combination is used as a vaccine. These vaccines are used to create more powerful combined immune

Question 1. I remember hearing in your video something about our body natural immunity and it eradicates the virus. The immune system includes physical barriers, such as the skin and mucous membranes that prevent pathogens from entering the body, and cellular responses, such as circulating macrophages that respond to foreign invaders. Our acquired immune system mounts a specific antibody response when the body is exposed to a foreign invader, and our immune cells attack the invader

Blood typing is a vital part of saving lives. People who have lost blood in any way will most likely need a blood transfusion. If the wrong type of blood is administered into their system, that person’s body will reject the new blood and the person will die. However, by knowing what antigens and antibodies are in a certain type of blood, the transfusion can be matched to the patient’s blood, which will lead to a safe, effective transfusion.Therefore, knowing what antigens and antibodies are in a person’s blood and what their blood type is can be a vital part of saving their life one day. For this reason, it is vital to know how antibodies affect blood typing.

The antigen-recognition molecules of T-cells are made as membrane bound proteins and function to signal T-cells for activation. These molecules are called T-cell receptors. T-cell receptors are similar to antibodies in structure, but they do not recognize and bind to the antigen directly. T-cell receptors will recognize short peptide fragments of pathogen protein antigens, that are bound to MHC molecules on the surfaces of other cells. Major histocompatibility complex, or MHC will present fragments derived from antigens and display them on the surface of antigen presenting cells to be recognized by the T-cell receptor. The TCR will recognize not only the peptide fragments of the antigen, but will also recognize the MHC molecules which the fragment is bound to, adding another dimension of specificity, relating to the analogy of a child trying to find its parent in a crowd.

In order for these events to occur forward and reverse oligonucleotide primers, nucleotides (dNTPs), and Taq polymerase must to be added to the PCR solution. The oligonulceotide primers complimentary to the target sequence and can be used as probes to detect the target sequence of DNA that will be used in PCR (Cox et al., 2012). The dNTPs act as building blocks for the new DNA strands that will be amplified and Taq polymerase is a thermostable enzyme used to create new DNA strands from the template (Cox et al., 2012)

Antibodies are used for many different purposes. According to WORLDPHARMACEUTICALFRONTIERS, 2014 saw $75 billion dollars spent on monoclonal antibodies which is more than half of the biopharmaceutical market 1. For this reason, novel methods of protein purification are needed to address the growing demand for antibodies. In this paper, the research group provides a novel method of purifying human immunoglobulin protein (IgG) by the use of a “repebody”.

Immunoglobulin (Ig) or Antibody (AB) is a protein that is in the shape of a Y that is produced by plasma cells and use by the immune system. These proteins are then utilized to identify and attack bacteria and viruses. Immunoglobulin is clearly a very important protein in our human bodies as well as being very integral to our survival and our ability to fend off foreign molecules. This has lead to a lot of deep research regarding this protein. By knowing the proteins exact structure and functions there will be a larger understanding of how our bodies actually work and how we can improve our health to live longer and better lives. This is exactly what will be looked at throughout this paper, the research, structure, function and behavior of the protein Immunoglobulin (Ig).