Acid Bit

By Brigette Federico

 Immunoassays such as Western blotting and ELISA often rely on a two-stage technique. A primary antibody (1o Ab) is used to bind directly to the protein of interest. A secondary antibody (2o Ab), labelled with a detection tag such as a fluorescent dye or colourmetric enzyme, is then applied. This binds to a site on the primary Ab, allowing indirect detection of the antigen.

Single stage assays have been developed, in which primary immunoglobulins are tagged, allowing both probing and detection to take place from the same molecule. However, the indirect approach is still preferred, for many reasons. One-step 1o Abs are custom-built and thus expensive. In addition, more than one 2o Ab can target the primary protein producing clearer results.

Primary Abs are typically purchased from antibody suppliers in monoclonal form. These are derived from a single cell-line and are totally homogenous. By contrast, most secondary proteins are polyclonal. Here, animals are injected with a specific peptide antigen of a particular species, a secondary immune response is triggered and in this way a whole plethora of proteins are produced unique for that antigen.

The difference between monoclonal and polyclonal proteins is that, while a monoclonal only recognises one specific epitope, polyclonals can recognise several. The primaries are raised against a specific antigen, and are unlabelled. 2o Abs are raised against primaries, and are labelled, or conjugated, with a reporter enzyme or one of the new fluorescent products such as Dylight.

Sometimes best results are obtained by using antibody fragments, such as an Fab fragment or gamma light chain, as the primary Ab rather than the entire protein. This can improve signal-to-noise ratio in assays, and improve binding to the antigen. The 2o Ab will be specific to Fab.

The secondary antibodies supplied by us at Novus Biologicals are available for a wide range of specie and primary proteins.

The Article is written by novusbio.com providing antibody database and antibodies Services. Visit http://www.novusbio.com for more information on novusbio.com Products & Services___________________________Copyright information

This article is free for reproduction but must be reproduced in its entirety, including live links & this copyright statement must be included. Visit novusbio.com for more services!

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Alternatives to embryonic stem-cell lines in antibody research

By Davis Morris

 Many suppliers include a range of stem cell marker proteins on their antibody databases. Marker antibodies, such as the one selective for Podocalycin-like protein (PODXL) are widely used in research into cancers and other diseases.

The PODXL gene has a similar structure to the CD-34 ligand, and encodes to a member of the sialomucin protein group. Originally identified on epithelial podocyte cells, it has been implicated in the development of aggressive tumours, and is used as a diagnostic marker in numerous cancers, including prostate cancer and pancreatic carcinomas.

At Novus Biologicals, our PODXL TRA-1-81antibody is one of a number of cell surface marker immunoglobulins designed for embryonic stem cell research. It recognises a specific carbohydrate epitope on the PODXL protein, the antigen TRA-1-81. Involved in cell differentiation, TRA-1-81 is expressed through PODXL on the surface of embryonic stem and germ cells, and also upon the surface of adult human teratocarcinoma stem cells. Cell surface antigens are known to play an important role in the ex-pression and function of developmentally controlled cells during embryonic and tumour development. For this reason, embryonic stem cells are routinely used in cancer studies involving TRA-1-81 and other PODXL immunoglobulins.

Since no murine (mouse) immunoreactivity is seen with our TRA PODXL antibodies, human cells must be used. The area of human embryonic stem-cell (hES) research is an extremely controversial one. However, in January 2010 Mizrak, Chikhovskaya et al, of the University of Amsterdam, released a paper suggesting an alternative human adult tissue. Studies had already shown that multipotent ES-type cells could be sourced from adult mouse testes. When these experiments were repeated using tissue donated from prostate cancer patients, subcultured cells expressed hES specific carbohydrate antigens as in the mouse studies. These included TRA-1-60 and TRA-1-81 both of which are on our antibody database.

This study means that human stem cell research is no longer restricted to embryonic cell lines an exciting development in an area which is historically extremely controversial.

The Article is written by novusbio.com/ providing antibody catalogue and antibodies Services. Visit http://www.novusbio.com/ for more information on novusbio.com/Products & Services___________________________Copyright information

This article is free for reproduction but must be reproduced in its entirety, including live links & this copyright statement must be included. Visit novusbio.com/ for more services!

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