The sandwich ELISA is a type of Enzyme-linked immunosorbent Assay that uses two antibodies: a capture antibody and a detection antibody. The purpose of any ELISA is to detect the presence of a target antigen in a sample. In a sandwich ELISA the target antigen is bound between a capture antibody and a detection antibody. The capture antibody is immobilized on a surface, while the detection antibody (conjugated to an enzyme or fluorophore label) is applied as a last step before quantitation.
ELISA stands for Enzyme Linked Immunosorbent Assay. The immunoassay utilizes the specific lock-and-key recognition between antibodies and antigens. This recognition occurs naturally in the adaptive immune system; antibodies are created by the immune system when an antigen such as a virus or bacteria invades the body. The immune system recognizes the foreign invader and creates antibodies that specifically recognize surface proteins on the virus or bacteria. The antibodies are either attached to the surface of an immune cell or move freely through the body to tag the invader and begin a cascade of destruction and elimination. The most useful part of this process from a biotechnology and engineering perspective is the specificity of the antibody-antigen recognition.
Filtration is a simple technique used to separate solid particles from suspension in a liquid solution. There are many filtration methods available, but all are based on the same general principle: a heterogenous mixture is poured over a filter membrane. The filter membrane has pores of a particular size. Particles larger than the pores will be unable to pass through the membrane, while particles smaller than the pores will pass through unhindered. Additionally, all liquids will pass through. The final result of a filtration process is a collection of residue on the filtration membrane. This residue is therefore effectively separated from the rest of the mixture that passed through the membrane.
Fluorescent microspheres are small polymers embedded with fluorescent dye. They are a useful tool for medical imaging because they are non-toxic and non-biologically reactive when used as directed. Fluorescent microspheres are also useful in research laboratories as markers for fluorescent microscopy and as standards for flow cytometry fluorescent cell sorting. The main benefit of using a polymer microsphere embedded with fluorescent dye rather than using the dye alone, is two-fold: the matrix protects the dye from photobleaching, and the microsphere concentrates the dye leading to a more robust fluorescent signal.
Biotinylation means attaching a biotin tag to a molecule. Biotin is a natural molecule that is also known as vitamin B7. It is an important component in a healthy diet, but it is also very useful in the laboratory in a method for protein conjugation. In the laboratory, he purpose of biotinylation is to create a controlled site for biotin-streptavidin affinity binding. Biotin and streptavidin have an extraordinary affinity for each other (Kd=10^-15). With such a low dissociation constant, once the biotin and streptavidin are bound it is unlikely that they will dissociate. This affinity is resistant to changes in temperature, pH, and salt concentration and is extremely specific. It is often thought of as a nearly covalent bond. These properties make biotinylation a useful tool for engineers who are developing new purification and detection methods. A commercially available biotinylation kit makes the process even easier.
The Enzyme Linked ImmunoSorbant Assay (ELISA) is the gold standard immunoassay for clinical diagnosis of disease. The basis of any immunoassay is the specific molecular recognition between antibody and antigen. This is something that the immune system does naturally. The production of monoclonal antibodies in a laboratory has become commonplace and standardized, which makes it possible to use monoclonal antibodies in immunoassays such as an IgG ELISA. The antibodies are easy to purchase from commercial vendors, and they come with quality control reports ensuring that they will recognize the target antigen.
Proteins are one of the four macromolecule building blocks of life. The other three are carbohydrates, lipids, and nucleic acids. Proteins are long strings of amino acids that fold together into hierarchal structures in order to perform specialized functions within the cells and tissues of all living organisms. These higher structures are imperative to the proper function of the protein within it biochemical pathway. The tertiary structure creates the chemical and morphological landscape that imparts the biorecognition abilities to ligand, receptors, antibodies, and all of the other workhorse proteins in the organism. The hydrophobicity or electrosatic nature of the binding pockets are responsible for the specific affinities between proteins. It is no wonder that the ability to analyze proteins with a protein assay is fundamental to biological research and clinical diagnosis.
Purpose of Protein Assays
The purpose of the protein assay is to determine the amount or concentration of a specific protein or an array of different proteins a sample. This can be a primary step before further manipulation in a research and development process, an initial capture of protein before structural analysis, or it can be a final detection step in a clinical laboratory as part of a disease diagnosis.
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.
Biodetection is a general term that encompasses the global strategies in place for the detection of biological threats. Biological threats are pathogens, infectious disease, and biological weapons that can infect significant populations of humans and to which we have little innate immunity or defense against. Wee must improve our ability to detect the infectious pathogens at the earliest sign of an outbreak. This will be accomplished by improving our methods of biodetection by developing more sensitive and portable biosensing devices. The use of bioassays in clinical laboratories are standardized and validated to improve the accuracy and speed of pathogen detection and disease diagnosis. New technologies are being developed to integrate biodetection platforms with smartphone devices and extend the sensing range to the hands of ordinary individuals.
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.