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) of the light chains. The L-chains are non-hinged, and sit inside the ‘arms’ of the Y.

The regions of polypeptide chains are called domains. The amino terminal end of each chain is termed the variable (V) domain. V-domains show considerable diversity compared to the C (constant) domains, and are where antigen binding takes place.

L-chains contain one variable domain VL, and one constant domain CL. The H-chains have one variable domain VH, plus 3 constant domains CH1, 2 and 3. The CH1 and CH2 domains sit either side of the hinge. Each H-L pair forms a single binding site, meaning each antibody unit is a bivalent monomer.

There are five primary antibody classes, and a number of sub-classes. Variations in the heavy-chain polypeptides and number of monomers allow them individual functions in the immune response.

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 becomes inactive if hypoxia occurs. The β-subunit is continually expressed.

HIF-α has three isoforms, known to be regulated by prolyl hydroxylation of the O2-dependent degradation domain, during the posttranslational phase. Prolyl hydroxylases target the protein via interaction with VHL (von Hippel-Lindau), one of the components of the E3 ubiquitin-ligase complex (VBC). VBC links ubiquitin to HIFα, tagging it for degradation by the cellular proteolytic proteasomal complex.

Additional hydroxylation takes place on the HIF1α and HIF2α C-terminus. This inhibits the binding of co-activators such as p300 and CREB-binding protein (CBP), thus inhibiting HIF-1 activation. The entire hydroxylation pathway is catalysed by 3-PHD (prolyl hydroxylase domain) isoforms, plus FIH (factor inhibiting HIF) proteins. Their activity is dependent upon cofactor Fe2+ , 2- oxoglutarate and oxygen.

Over-expression of PHD-3 is associated with aggressive pancreatic tumours, while p300/CREB studies have shown similar results. Therefore HIFα antibody studies continue to be important in cancer research.

Each week, our lab staff at Novus Biologicals produce, conjugate and purify hundreds of products for our antibody catalogue. Purification protocols are specific to each antibody. For example, we recently purified rabbit polyclonal anti-OCT4 to a final concentration of 1.2 mg/m, using a peptide affinity column. We also purified 20ml of c-Myc ascites over a protein G column, to create purified mouse mouse monoclonal anti-c-Myc (9E10) antibody to a final concentration of 0.82mg/ml.

Improved immunoassay technology means antibodies must be extremely pure, with minimum risk of false positives and “noisy” results. A wide range of purification methods exist to ensure this is possible. Which one we use depends on the Ab class, the species it was raised in and its intended use.

Antibodies are prepared from serum, tissue culture supernatant or ascites (peritoneal fluid containing immunoglobulins) and then purified by one or more of the following methods: Protein A/G purification; Gel filtration; Ammonium sulphate precipitation; Ion exchange chromatography and immunoaffinity purification. The final product is supplied with a full protocol sheet and QC report stating yield, purity and immunoreactivity analysis.

Affinity column immunopurification is commonly used to produce antipeptide antibodies. The peptide is first immobilised on sepharose beads, and the serum is then loaded onto an affinity column. The bound antibodies are eluted, and ELISA is performed on both the immunopurified Abs and the crude serum.

Antibody purification on a protein column (A or G) is similar, but following ELISA the proteins undergo further SDS-PAGE analysis. This yields 2 – 4 mg/ml of serum. Another purification method is IgY purification from egg yolk extract, on an ion exchange column.