Scientific Strategy

About Antibodies

LifeSpan’s goal is to provide high-quality antibodies that target every therapeutically relevant protein target in the human body. We currently offer one of the industry's largest catalogs of monoclonal and polyclonal antibodies covering virtually every protein class and all applications. Many of our antibodies have been characterized for use in immunohistochemistry!

Antibody Generation and IHC Validation

LifeSpan offers the industry's largest inventory of rabbit polyclonal anti-human peptide antibodies to gene families including the G Protein-Coupled Receptors (GPCRs), Nuclear Receptors (NRs), and Kinases. LifeSpan's antibodies have been optimized for use in human tissue immunohistochemistry. The expression pattern of each antibody has been screened across a panel of at least 50 tissues and compared to other proprietary or commercially available antibodies. Antibodies are selected for those that show the strongest concordance to existing literature and known patterns of expression in other species, and for their affinity and specificity to their targets. Many of these antibodies are multi-functional and can be used in other assays such as cell sorting or Western blots.

LifeSpan has optimized its immunohistochemistry (IHC) protocol and antibody selection for the detection of low-abundance proteins in formalin-fixed, paraffin-embedded tissue specimens. These samples afford the best morphology and represent the broadest possible range of tissues and diseases.

LifeSpan’s antibody generation protocol begins with the synthesis of peptides based on non-overlapping regions of the target protein. Peptides are selected to be as unique as possible to the target protein and to have minimal homology to closest homologues and to other proteins in the human genome. Each peptide is conjugated to keyhole limpet hemocyanin (KLH) and is used to immunize rabbits. Sera from the immunized rabbits are tested for binding to the cognate peptides by ELISA (enzyme-linked immunosorbent assay). Sera are then purified on a peptide-affinity column and are then re-evaluated by ELISA. Multiple antibodies to each protein are used to immunolabel multi-tissue sections that contain up to 50 human tissue types, including known positive- and negative- control tissues. The results are analyzed by our anatomic pathologists, and the staining patterns produced by the antibodies are evaluated for cross-concordance with each other as well as with the published literature.

LifeSpan has established staining criteria that must be met before an antibody is considered to be both specific for its target protein and suitable for use in IHC. For receptors that have been studied previously, the pattern and anatomic distribution of staining should be consistent with what has been reported in the literature. Supporting evidence from published sources may include immunohistochemistry, Northern-blot analysis, in situ hybridization (ISH), EST (expression-sequence tag) expression profiling, radioimmunoassay, and ligand-binding data. Data from other species are also evaluated (i.e., rodent brain ligand binding) and compared to the staining pattern obtained with each antibody.

For orphan receptors, supporting data from binding studies and ligand-mapping studies are not available, and evaluation of the specificity and validity of our IHC findings relies more heavily on cross-concordant findings with multiple antibodies and on gene expression studies such as Northern-blot analysis or in situ hybridization. Generally, proteins that are detectable within a particular type of tissue by Northern-blot analysis are also detectable by IHC. Although it is possible for a gene to be transcribed within a tissue but not translated, our experience has been that a positive finding on a human tissue Northern blot generally correlates with the ability to detect the protein within that tissue by IHC. Northern analysis can, however, underestimate the presence of low-copy mRNAs or those that are expressed by only a small subset of cells within a tissue. In situ hybridization (ISH) is a more sensitive method for the detection of low-abundance genes, because mRNA can be localized to individual cells within a tissue sample. However, this method does not lend itself readily to high-throughput, first-pass screening in formalin-fixed, paraffin-embedded tissue specimens, and many cell types may produce mRNAs that are below the limit of sensitivity of detection by colorimetric or radiometric ISH.

Another criterion for evaluating antibody specificity and performance is that the subcellular localization of the staining signal should reflect the protein's function. Depending on the specific protein, the size of the labeled cell compared to the section thickness, and whether the cell has been transected in the section, labeling may be concentrated on the periphery or appear to completely fill the cell, or be primarily located in the nucleus.

For an antibody to be suitable for use in IHC, it should show a good signal-to-noise ratio (e.g., specific staining should be considerably higher than the level of nonspecific background staining) on tissue specimens. High-affinity antibodies produce the strongest, most reliable data, because the affinity of an antibody for its target protein affects its sensitivity and its specificity.