Blogs for February 2010

Not everybody working in immunobiology has an in-depth knowledge of the subject. Some may be students, who are still getting to grips with the discipline at college. Others may have been forced into a swift career change following restructuring at work. For whatever reason, people quite often perform their first antibody assays with only the vaguest knowledge of the underlying concepts. Understanding the molecular structure of a given antibody is fundamental to interpreting its results, therefore we at Novus Biologicals have put a few basic facts together.

Antibodies are glycoproteins composed of one or more Y-shaped polypeptide units. Each of these has two identical heavy (H) and two light (L) chains, forming the left and right binding sites of the Y. The H chains are hinged, and have roughly double the number of amino acids (and therefore molecular weight)...

IgG is the most prevalent antibody in mammalian tissue, and therefore a major number of the proteins on an antibody database are of this type. The other classes that are studied are IgA, IgM, IgD and IgE. Depending on the tissue and disease being studied, polyclonal and monoclonal versions of all these antibodies are also produced against specific antigens. Cross-reactivity can occur in IHC assays using tissue-derived antibodies; therefore it is common to use fragments, rather than entire Igs as primary immunoglobulins.

Antibody classes are differentiated by their heavy chain structure. The light chains are fairly homogenous, but minor differences can occur among the H-chains of some classes. Therefore these are further divided into sub-classes. IgG has 4 such subclasses. However,...

Many people enter immunobiology from a different area of research. Although they swiftly learn the protocols and methods of the assays, they may not fully understand the underlying concepts. We at Novus Biologicals realise that not everybody purchasing products from our antibody catalogue has an in-depth knowledge of the “tools of the trade”. For them, and anybody else needing a quick bit of revision, we have put together a few basic notes.

Most people quickly understand that antibody proteins are produced by B lymphocytes, and are specific to foreign proteins (antigens) that enter the body. The body’s natural ability to produce such antibodies has been utilised into a useful research tool. Synthetic and natural antigens are used to generate antibodies, which are then used as probes to detect and bind to those target...

Recently, we at Novus Biologicals have added a new batch of monoclonal anti-HIF-1 alpha antibody to our hypoxia catalogue, developed from H1alpha67 hybridoma cultures. This furthers the role that HIF-1 antibodies play in cancer research.

HIF-1 becomes active under hypoxic conditions. Its function is the transcription or blocking of a number of genes, to preserve cell viability at times of low oxygen stress. However, HIF transcription can also be activated in non-hypoxic conditions, and HIF expression has been shown to be a factor in tumour development. Therefore both normal and cancerous cell-lines are used in HIF-1 antibody studies.

HIF-1 is a heterodimer complex composed of identical β and α subunits, which must both be expressed for HIF-1 to become active. Under normoxic conditions, the α-subunit is degraded by the cellular proteasome, a process that...

A growing number of studies are looking at causes of cancer at a molecular level – and discovering that hypoxia pathways play a major role. This is because HIF (Hypoxia Inducible Factor) proteins work to prevent cell death. Combined with DNA-repair pathways and the apoptosis mechanism, it’s obvious what disruption of these pathways could lead to.

Antibody studies have focussed on hypoxia-induced autophagy. The HIF-1 pathway is linked to BNIP3 and BNIP3L release, pro-apoptotic genes expressed in certain tumours. It has been shown that BNIP3 expression during hypoxia may result in mitochondrial autophagy, rather than whole-cell death. Thus BNIP3 may play a cell-survival role, decreasing dependency for oxygen. If DNA-repair is compromised, this may lead to cancer development.

Studies into the pRB/E2F protein complex have shown high levels of activity in hypoxia-induced autophagy (HIA) when activated in cancer cells. pRB/E2F has also...

Hypoxia-inducible factor (HIF) is a DNA-binding protein that regulates homeostasis via transcription of a wide range of genes. It is inactive in oxygenated cells, but becomes active in hypoxic, i.e. low oxygen conditions. HIF proteins consist of a heterodimeric complex of identical alpha and beta sub-units. The α is degraded in normal oxygen conditions, while the β remains independent of O2 concentration.

HIF is implicated in a wide range of tumours. Recently, we at Novus Biologicals cultured the H1 alpha67 hybridoma cell line to produce the monoclonal anti-HIF 1 alpha antibody. This is one of a number of products in our hypoxia antibody catalogue routinely used in cancer research.

The involvement of HIF-1 in tumour development is obvious when you consider the complexity of the hypoxia pathway. Under normoxic conditions, the α-subunit undergoes prolyl hydroxylation and is tagged by E3 ubiquitin ligase for degradation by the cell...

All cancer genomes carry somatic mutations. These include base substitutions, rearrangements, insertions and deletions (indels). Antibody suppliers have seen tremendous growth in the field of cancer research recently, with antibodies to at least 400 cancer genomes.

Driver mutations are direct somatic changes which confer an advantage for clonal growth and can lead to oncogenesis. Passengers or “silent mutations” do not lead to cancer development, but give important information on pre-cancerous cell changes. Epigenetic mutations do not alter the genetic code, but can still be passed on to at least one successive generation. All are of interest.

DNA damage, such as strand breaks, is common in normal cells, and most do not cause mutation. The changes are recognised by cell enzymes, and various DNA repair mechanisms, such as NER (nucleotide excision repair) correct the damage and prevent driver...

Antibody suppliers have seen an enormous growth in the area of DNA repair in recent years. DNA damage causes structural changes within the cell that can cause genome mutations to occur, a common cause of tumour development and an important area of research for antibody suppliers like us at Novus Biologicals.

NER (nucleotide excision repair) is a global genome repair process, activated by DNA damage caused by (among other things) UV light. This is a common cause of malignant melanomas. NER repairs distortions of the DNA helix by excising the oligonucleotide (short strand section) containing the lesion. Reproductive DNA polymerases fill the resultant gap using the undamaged strand as a template. There is also a transcription-coupled form of NER, which prevents modification of the transcription process by damaged DNA. This appears to be more efficient at preventing mutations, which usually occur at...

Research facilities often require a supply of antibodies of all classes, in both their whole and fragmented forms. There are both pros and cons to using fragments in place of entire proteins. As IgG is the most prevalent class on our antibody database, we’ll cover that one first.

IgG consists of two heavy chains and two light chains, held together by hydrophobic interactions and disulphide bonds. Secondary (anti-IgG) antibodies are derived by injecting an animal of one species (e.g. a sheep) with whole Ig from another species (e.g. mouse) that produced the 1° Ab to the antigen. This results in polyclonal secondary Ab which reacts to both the H and L chains. It reacts to every possible primary epitope, resulting in the largest possible signal.

However, the same light chains are common in all the antibody classes,...

FANCD2 is one of a number of proteins in the FANC group. It undergoes modification in both its normal and disease state; therefore our FANCD2 antibodies are modified and conjugated in various ways for research.

FANCD2 (Fanconi Anaemia Complementation Group D2) is one of several FANC proteins involved with development of Fanconi Anaemia, a genetic disease that increases cell susceptibility to tumour development. In its normal pathway it interacts with several other genes known to cause cancer, for example BRCA1.

FANCD2 is routinely modified by interaction with other proteins, to enable it to carry out normal tasks. The protein occurs in all cells and plays a role in DNA-repair, inhibiting DNA synthesis following exposure to ionizing radiation, as well as resisting cross-linking. It also activates S-phase checkpoints following phosphorylation by ATM. It has been shown FANCD2 may also be part of the DNA repair mechanism, repairing...