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.
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.
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.
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:
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.
Faster and more efficient methods of pathogen detection are in high demand. The traditional methods involve collection of patient blood or swab samples for multi-day cultures. These methods are time-consuming and require full laboratories with skilled technicians and sterile equipment. As such, they are not ideal for low-income areas or for rapid pathogen detection. There is a need for rapid pathogen technology and point-of-care diagnostic tools. Ideally, these technologies will come with a built-in validation protocol. Magnetic nanoparticles and molecularly imprinted polymers are good candidates for improved pathogen detection systems. An additional benefit to using magnetic nanoparticles is that the separation process is easy to quantitatively measure with a validation protocol.
The northern blot protocol is a technique used to study gene expression via mRNA transcripts. The northern blot was named after the southern blot, which was developed to study DNA. The two techniques are the same except that the northern blot is used to detect RNA while the southern blot is used to detect DNA. The northern blot protocol, in brief, involves gel electrophoresis to separate mRNA by size, a blotting step to transfer the separated mRNA to a membrane, and a probe hybridization step to identify the mRNA sequence of interest. Even with the advent of powerful RNA analysis techniques such as RT-qPCR and sequencing, the northern blot protocol is still useful for comparing gene expression between samples. The northern blot protocol is relatively inexpensive, and makes it easy to visualize the results on a single membrane.