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.
Fundamental research often involves the study of isolated cell populations. It is these enriched populations that enable researchers to make new discoveries about cell function, signaling, gene expression, fate decisions, and much more. Techniques for the rapid and accurate enrichment of target cell populations are an area of great interest. Cell sorting techniques fall into two general categories: bulk sorting and single cell sorting. In bulk cell sorting all of the target cells are collected in one sweep, whereas in single cell sorting every cell is individually analyzed. There are multiple methods of bulk cell sorting: filtration, centrifugation, and magnetic cell sorting. The main single cell sorting method is flow cytometry or fluorescence activated cell sorting. While cell sorting can be very accurate, it is hard to say that a sorted cell population is “pure”. Instead, the collected population is referred to as “enriched”. In general, single cell sorting results in highly enriched cell populations that are more homogeneous than those obtained via bulk sorting methods.
Molecular diagnostics entails the analysis of biomarkers to help diagnose, track the progression of, or determine risk factors and prognosis of disease. Biomarkers have been identified within the realm of genomics, epigenomics, transcriptomics, proetomics, metabolomics, and lipidomics:
The ELISA or Enzyme-Linked Immunosorbent Assay is the most established method of protein detection and quantification. The technique is commonly used in a wide variety of applications spanning from the basic research bench to clinical laboratories. The ELISA is a labeled assay, which means that it requires the use of a label to detect an antibody-antigen binding event. The label is commonly a fluorescent probe, chemiluminescent system, or enzymatic colorimetric reaction.
Molecularly imprinted polymers (MIP) are relatively new diagnostic and therapeutic tool. MIPs are highly specific three dimensional polymer imprints of molecules. The power of this tool may not be immediately obvious, but it is indeed a very useful technology. The power of MIPs lies in their specificity of binding to target molecules. Before the invention of MIPs, the only way to achieve this type of specificity was through antibody-antigen binding, surface receptor-ligand interactions, or protein affinities such as streptavidin and biotin. The problem with all of these systems is that they need to already exist. But what about a target that doesn't have a known molecule with a natural affinity? Herein enters molecularly imprinted polymers. These polymers are crosslinked and formed around the target molecule. Once the polymerization process is complete, the target molecule template is destroyed. What remains is a three-dimensional polymer with binding sites in the exact physical and chemical configuration as the target molecule. These binding sites are specific with regard to hydrophobicity, hydrophilicity, chemical groups, hydrogen bonding, and charge. When these MIPs are added to solution they will bind the target molecule with exceptional specificity.
For the indirect and direct elisa, the antigen is applied to the surface of the elisa plate, but for a capture antibody is attached directly to the surface of the plate for sandwich elisa. Aside from this major difference the indirect and elisa protocol is very similar to the sandwich elisa protocol. There are plenty of blocking and washing steps to avoid non-specific binding, and there are incubation times to allow antibodies and antigens to bind properly. The indirect elisa requires two antibodies—a primary antibody to bind to the antigen, and a secondary antibody conjugated to an enzyme or fluorophore. The direct elisa uses a primary antibody that is directly conjugated to an enzyme or fluorophore. Either way, both of these methods—and indeed every elisa protocol, is a labeled assay. The antibody-antigen binding event cannot be quantified without the presence of the enzyme or fluorophore.