Antibodies are naturally produced by the adaptive immune system in response to invading pathogens. The antibodies are made by immune cells to specifically recognize protein markers called antigens located on the outer wall or membrane of the pathogenic organism. It is this exquisite antigenic specificity that makes the adaptive immune system so remarkable in its ability to fight off a wide variety of diseases. It is also this specificity that makes the antibody-antigen interaction an attractive tool for the development of biological assays for the detection of active infection and disease.
The BCA protein assay is used to quantify total protein in a biological sample. BCA stands for Bicinchoninic acid, which is the key reagent used to produce a colored product. The purple colored product is analyzed in reference to a standard curve in order to quantify protein concentration. It is important to measure protein concentration after performing a protein extraction or purification, and prior to any type of labeling procedure. The protein concentration after extraction or purification may provide information about a biochemical pathway or a disease state. All commercially available proteins are accompanied by a product information sheet that has the results of a protein quantification method. This is particular important in antibody validation. It is important to know the protein concentration prior to any labeling step so you can ensure that the stoichiometric ratio between label and protein is optimal for clean and efficient labeling. It is equally important to know how much protein you are working with when designing biosensors so that you can define limits of detection and instrument sensitivity.
Antibodies are a key component to many biotechnical applications. They are most often used for immunoassays such as ELISA, cell and tissue staining, protein quantification such as western blot, and cutting edge sensor development. Verified antibodies are easily purchased from commercial vendors. These antibodies can be monoclonal or polyclonal, and can come as a lyophilized powder or as a premixed solution. All of these details must be considered when choosing which antibody to purchase because they all have an effect on the antibody concentration and dilution process.
Protein Purification is essential to understanding the structure and function of various proteins. It is also important for the development of clinical biosensors, which rely on stocks of pure protein as detection agents. There are many methods of protein purification, which are discussed below along with how protein purification works, the steps involved, and how to increase yield in your protein purification protocol.
The ELISA, or enzyme linked immunosorbent assay, is the gold standard immunoassay for detection of small quantities of protein in samples as varied as serum, urine, saliva, and more. The ELISA is a labeled assay, which means that some type of label is needed to detect protein binding events. These labels are typically fluorescent, chromatic, or chemiluminiscent, and require the use of a plate reader to quantify the amount of protein in the sample. The major benefit of the ELISA is that low concentrations (often down to pg/mL) of protein are easily quantified. One disadvantage to ELISA is that many steps and reagents are required throughout the protocol. However, this can be mitigated by purchasing an ELISA kit that is pre-bound with capture antibodies and contains a detailed protocol for using all of the included buffers in a clear, easy to follow format. The use of an ELISA kit can improve diagnostic results from assay to assay because the kits are all validated between lots and come with protein standards. This means that a standard curve (detected signal vs. protein concentration) is generated during each assay and this standard curve can be checked against the expected values to ensure that the kit is still functioning as expected. The kit streamlines the process and takes the guesswork out of protocol design.
Antibodies are produced by the adaptive immune system in response to invading pathogens. The antibody has specific lock and key recognition for the offending bacteria, virus, or other molecule, which are collectively called antigens. Antibodies are proteins, which are folded polypeptides, or strands of amino acids which have antigen recognition sites that specifically recognize a binding site of its specific antigen. They are produced by B-cells of the adaptive immune system.
Proteins are fundamental building blocks for life. All tissues and organisms are made up of protein, and all of the work performed inside and outside of cells is mediated by protein signaling cascades. Proteins are polymers of amino acids with three or four layers of organized structure. Primary structure is defined as the linear order of amino acids. This is dictated by the genome: the code is transcribed from DNA and translated into the string of amino acids. Secondary structure is thought of as two basic forms: a beta sheet or alpha helix. The string of amino acids adopts the conformation that allows the lowest energy state. Beyond the sheets and helices, the chain can take other twists and turns to fold into a shape known as its tertiary structure. Some proteins are actually made up of two or more subunits of individually folded amino acids strands. The complexing of protein subunits to form one functional protein is called quaternary structure. All of this folding is extremely important to the character and function of each individual protein because it results in certain side chains of amino acids being located on the exterior or interior of the protein. Importantly, the folds create binding pockets where key amino acids are located to create a unique chemical landscape that allows the protein to bind to other proteins and carry out its job in a signaling cascade.
An antigen is defined as anything that causes an immune response in another organism. This immune response can be a simple increase of inflammatory factors, or it can be an activation of the adaptive immune system and creation of antibodies. Antibodies have two or more specific paratopes, or antigen recognition sites, that identify and combat the invading antigen. The number of antigen recognition sites is dependent on the antibody class. The word “antigen” can also refer to any protein of interest detected by a bioassay or biodetection platform. In the case of a bacterial antigen, we are referring to surface proteins, lipopolysaccharides, and peptidoglycans on the bacterial cell wall; these structures help bacteria invade other organisms by gaining access between epithelial cells. While surface structures help bacteria infect other organisms, they are also a detriment to the bacteria because they also serve as a unique tag that antibodies and bacteriophages can recognize. Bacteriophages are viruses that attack bacteria. Both antibodies and phages are being used by scientists to develop new biodetection and biosensing platforms for rapid detection of bacterial antigens in the environment and in clinical samples.
Protein purification services are available for anyone who is in need of a custom antibody or recombinant protein for research and development purposes. If your laboratory is not equipped to produce recombinant proteins in house, then this may be an attractive option. These services require you to provide a sequence and preferred expression system; in turn they will deliver a quality controlled protein with accompanying documentation to you within just a few weeks.