Novus Biologicals | Antibody News

ELISA is a widely used technique for detecting concentration of proteins, using antibodies tagged with enzyme which react with dyes to produce a colorimetric or fluorescent signal. Sandwich Elisa takes this one step further, by pre-coating plastic wells with a known concentration of a “capture” antibody which reacts specifically to the antigen under test. A secondary antibody is then applied, tagged with an enzyme.

Recently, we at Novus Biologicals added a range of Biosensis ELISA kits to our antibody catalog, covering a wide range of antigens important to human disease research. Among them is human BDNF – a protein which was recently in the news as it appeared to have a stress-reducing and possible tumour-reducing effect in cancerous mice subjected to exercise.

BDNF, or Brain-Derived Neurotrophic Factor, is a member of the neurotrophin growth factor (NGF) family of proteins. Its function is to regulate neuronal differentiation and survival during embryonic development, and to regulate the plasticity and transmission of CNS synapses in the adult. Its expression is affected by stress, seizures, hypoglycaemia and other conditions, and it has been linked to CNS disorders like Alzheimer’s, depression, epilepsy and Parkinson’s disease.

Our human BDNF sandwich ELISA kit is a highly sensitive, reproducible method of determining BDNF levels in a range of sera. It contains a strip-plate of 96 wells, pre-coated with polyclonal human BDNF capture antibody. Addition of test sera creates an antibody-antigen complex. Biotinylated BDNF monoclonal detection antibody is then added, which also binds to the antigen (hence the “sandwich.”) ABC (Avidin-Biotin-Peroxidase) enzyme complex then binds to this second antibody. Finally, TMB peroxidase substrate reacts with the ABC component to create a detectable coloured dye reaction, the intensity of which is proportional to the concentration of BDNF in the samples.

The best antibody suppliers offer far more than just individual peptides and reagents. They also supply a range of antibody kits which contain everything the scientist needs to perform a particular experiment in one convenient package. We at Novus Biologicals have an extensive range of such kits, including purification, labelling, epitope tag, ATPase and ELISA kits. Recently, we added Biosensis 96-well ELISA kits to our antibody catalog. These are considered to be the most sensitive of all ELISA kits, offering a low number of blanks and highly reproducible results.

ELISA (enzyme-linked immunosorbent assay) is a standard immunological technique used to detect the presence of an antigen in a sample. An antibody targeted to the appropriate antigen is first fixed to a plastic surface (i.e. a well) and a solution containing an unknown quantity of the antigen is then applied. This forms a quantifiable antibody-antigen complex. EIA (enzyme immuno assay) is a similar technique, except in this case a known concentration of antigen is fixed to the wells, and the quantity of antibody in a titer determined.

To make the ELISA antibody-antigen reaction visible, a detectable marker must be present. Generally, a secondary antibody (AB2) is used, targeted to a specific region of the primary antibody, (AB1). The AB2 is coupled to an enzyme which, in the final part of the process, converts an applied substance to a detectable signal, for example fluorescence or a dye. The intensity of the signal is equal to the amount of antigen present.

As you can see, the nature of the ELISA technique makes it ideal for supplying in kit form. Once the biologist knows the antigen they want targeted, practically any antibody can be supplied in pre-fixed well format.

Recently, we at Novus Biologicals added a new phospho-MAP1B (phosphorylated microtubule-associated protein 1B) antibody to our antibody database. MAP1B is a developmentally regulated phosphoprotein thought to be involved in the assembly of microtubules, an essential part of neurogenesis.

Gene knockdown antibody studies have shown MAP1B plays an important role in the function and development of the nervous system. It may regulate microtubule function, affecting neuronal differentiation and migration, axon growth and growth-cone function. Mutated MAP1B has been implicated in a range of neural disorders including Fragile X syndrome and giant axonal neuropathy.

Antibody studies have shown MAP1B is phosphorylated by GSK3 beta (glycogen synthase kinase 3beta) at two sites – Thr1265 and Ser1260. Phospho-MAP1B antibody (also – curiously – known as SuperBUGS) detects phosphorylated MAP1B at Thr1265.

Recent studies revealed phosphorylation cannot occur at the GSK3 beta site without pre-phosphorylation at a “primed site” further downstream. Primed GSK-3beta-phosphorylated MAP1B sites are found uniformly throughout the neuron, including the soma and axon. However, there are also a number of non-primed sites, which are only expressed in the axon area. These are arranged in a gradient that is highest towards the distal, or growth-cone end.

At both primed and non-primed GSK-3beta sites, phosphorylated MAP1B occurs in spatially distinctive patterns. Recently, Tymanskyj et al used antibody assay techniques to investigate the evolutionary conservation of MAP1B spatial distribution in embryonic spinal cord sections.

Phospho-MAP1B antibodies raised to both primed and non-primed GSK-3beta sites in a variety of embryonic vertebrates revealed both types were remarkably conserved. However, the lowest degree of conservation occurred in non-primed sites, suggesting these evolved more recently.

Recently, we at Novus Biologicals added several embryonic stem cell marker products to our antibody catalog, validated for use in fluorescent activated cell sorting (FACS) assays. They included Cripto1, PODXL, SSEA, OCT4, Nanog, SOX2, TRA-1, TERT and GPR49/LGR5 antibodies.

FACS is a type of flow cytometry which allows the sorting of a heterogeneous mixture of cells into individual containers, one cell at a time, based upon the fluorescent and light scattering properties of each cell. It is useful characteristics of each cell. It is a useful technique, providing a fast and quantitative recording of individual fluorescent signals, as well as physically separating cells of interest.

The technique is of particular use in stem cell research. When cells are obtained from multicellular organisms, the DNA is naturally identical in each one. However, the proteins of each cell vary widely. Therefore, a method of separating cells based on their phenotype i.e. FACS is extremely useful. In addition, FACS allows us to see what percentage of the total cell population is expressing the protein of interest, and in what quantity.

The process involves the gentle forcing of cells through a fine, vibrating nozzle, one at a time. As the cells flow through, they are scanned by a laser. Some of the light is scattered, and this is used to count the cells. It also serves to measure the size of the cells.

Cell subpopulations can be separated by tagging with an antibody conjugated with a fluorescent dye, targeted to a protein specific to those cells. When excited by the laser, the dye emits a particular wavelength of light, allowing the apparatus to identify and isolate the relevant cells.