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.

LifeSpan's immunohistochemistry services provide high-quality information about the localization of proteins in normal and pathologic tissues. For each protein studied, optimal antigen retrieval and IHC labeling conditions are identified, the tissues are immunostained and analyzed by an anatomic pathologist, and the results are presented through an online client interface. All custom protein expression-profiling studies are confidential to the customer and are performed on a fee-for-service basis without royalties or milestone payments.

Study Design: Each contract research study begins with a customer consultation to define the study goals, available resources, and the most optimal approach. LifeSpan has conducted hundreds of localization studies for more than 100 companies worldwide, experience that can be applied to help ensure a successful study.

Antibodies: Antibodies can be provided by the client, purchased from a commercial resource, acquired from LifeSpan's antibody inventory, or custom generated by LifeSpan.

Tissue Selection: Customers work directly with LifeSpan to select tissue specimens or tissue microarrays that will best answer their biological question of interest. LifeSpan has over two million specimens in its tissue bank and can, in most cases, satisfy very specific parameters for analysis. Tissues can be animal or human, supplied by the client, acquired from LifeSpan’s extensive human tissue bank, selected from LifeSpan’s inventory of tissue microarrays, or collected prospectively by LifeSpan for the client. LifeSpan also offers custom tissue microarray design. Studies are then divided into two phases.

Phase I: Antibody and Assay Optimization
Customers can use their own antibody, LifeSpan's antibodies, commercial antibodies, or generate proprietary antibodies with LifeSpan’s antibody production service. LifeSpan subsequently optimizes the assay conditions for the antibody with the use of specified positive- and negative- control tissues. Optimization involves performing a serial dilution of the antibody to establish the optimal staining concentration and may involve optimizing antigen-retrieval conditions to improve the antibody's signal-to-noise ratio on tissue samples.

Phase II: Analysis and Reporting
LifeSpan tests each selected tissue with control antibodies to ensure that antigen preservation requirements are met. The experimental antibody is then used under the optimized assay conditions to immunostain the tissues of interest. The resulting localization pattern is then interpreted by a LifeSpan anatomic pathologist and digital photomicrographs are taken to demonstrate the localization that is reported by the pathologist.

Deliverables: Upon the conclusion of each study, LifeSpan will provide a report detailing the assay condition, the antibody dilution specifications, and localization results for each tissue specimen assayed. Selected patient information, including the age, sex, cause of death or surgery, is provided with each sample. LifeSpan offers a variety of reporting formats to meet the varied needs of our customers. Reports are delivered via secure website and can also be delivered on CD.

Full-Text Reporting: With full-text reporting, LifeSpan pathologists describe the immunolocalization patterns in detail and provide 3-5 representative images for each tissue sample. This format is useful for studies that require detailed observations and interpretation, such as studies involving CNS tissues, or co-localization studies, or those that may involve rare events and minor cell types. Information about each patient sample is also provided.

Tabular Reporting with and without Imaging: With tabular reporting, LifeSpan’s pathologists quantify the signal present in major cell types within each tissue sample; in a 0-4 scale and present the results in a table. This format is useful for studies at the initial stages of target characterization, screening studies, or those involving major cell types or in homogeneous tissues. Customers can choose to include selective images for each tissue sample.

Timeline: Standard IHC studies take from 3-12 weeks to complete, depending upon the complexity of the study. LifeSpan strives to complete our customers' work in an expeditious manner.

Cost: LifeSpan offers unparalleled quality and competitive pricing for its contract research services. To request a price quote, contact your local LifeSpan Sales Representative.

With more than 2 million samples, LifeSpan has the largest on-site collection of normal and diseased human tissues in the industry. Virtually every type of normal tissue, disease, and stage of disease is represented, and each specimen is validated for accurate pathology, quality fixation, and antigen preservation prior to use. These tissues are used in the development of LifeSpan’s localization databases and are also made available for custom designed contract research studies. This unrivaled resource provides LifeSpan with the range of samples necessary to pinpoint gene expression specific to a given disease, down to a particular stage of that disease. Our tissue bank is comprised of tissue samples from all major organ systems. It includes samples representing more than 1,500 diagnostic categories. LifeSpan does not sell tissues outside of tissue microarrays at this time.

LifeSpan's anatomic pathologists are available on a fee-for-service basis to analyze human or animal tissue specimens provided by our customers. LifeSpan specializes in the interpretation of IHC and in situ hybridization slides and has experience with over 4,000 antibodies to the major drug target families, and has provided confidential data to over 100 customers worldwide. LifeSpan can provide high-throughput tabular reports, comprehensive pathology interpretations, and high-resolution digital images. LifeSpan's pathologists have expertise in all aspects of general surgical pathology, including neuropathology, cytology, cancer pathology, lung pathology, dermatopathology, cardiovascular pathology, reproductive pathology, and renal pathology.

LifeSpan has generated and is continually expanding the industry's most comprehensive gene expression and protein localization data resource, the LifeSpan DrugTarget Database^TM. This relational database features extensive proprietary immunohistochemical data for many pharmaceutically important proteins. The localization profiles for more than 380 proteins have been studied across the normal human body, including most peripheral tissues, the mapped brain, and more than 20 diseases of major therapeutic interest. Acting as a backbone for this database is LifeSpan’s informatics suite, DrugTarget Portal^TM, which contains data curated from the public domain for more than 3,200 genes across eight therapeutically focused gene families, including the G protein-coupled receptors, ion channels, protein kinases, nuclear receptors, protein phosphatases, phosphodiesterases, proteases, and transporters. Portal delivers comprehensive, hand-curated expression data through a unique and intuitive interface, allowing for the rapid retrieval of relevant expression information.

LifeSpan’s databases include innovative tools that enable subscribers to rapidly locate information relevant to their own interests from both the public domain and LifeSpan’s proprietary localization studies. Criteria such as gene name, synonyms, sequences, celltype, tissuetype, assays, expression results, and disease association can be used to sort, filter, and organize the data in a meaningful way. Subscribers find our databases to be invaluable assets to their drug-discovery programs.

For more information or access to a FREE database demonstration site, contact your local LifeSpan Sales Representative.

LifeSpan uses multiple antibodies to study the localization profile of each gene across an extensive panel of normal and diseased human tissue types. Each antibody, selected for its specificity and reactivity when used under LifeSpan’s standardized assay conditions, is used to immunolabel two patient specimens each of at least 25 normal peripheral tissue types, 11 normal brain regions, and 25 diseases of major therapeutic interest. An anatomic pathologist interprets the immunolabeling results, which are then reported along with multiple images of each tissue type.

Normal tissues are studied from the following list:

Adrenal	Liver	Skin
Artery	Lung	Spleen
Bladder	Lymph node	Stomach
Breast	Muscle, skeletal	Synovium
Bronchus	Nasal mucosa	Testis
Colon	Ovary	Thymus
Heart	Pancreas	Thyroid
Intestine	Placenta	Uterus
Kidney	Prostate

Brain regions are studied from the following list:

Amygdala	Hippocampus	Putamen
Caudate	Hypothalamus	Substantia nigra
Cerebellum	Medulla	Thalamus
Cortex	Pituitary

Tissues with the following disease diagnoses are studied:

Artery, Atherosclerosis	Lung, Emphysema
Brain, Alzheimer's disease	Lung, Non-small cell carcinoma
Brain, Glioblastoma	Lung, Pneumonia
Brain, Infarct	Lung, Small cell carcinoma
Brain, Parkinson's disease	Lymphoma, Hodgkin's disease
Breast, Carcinoma	Lymphoma, Non-Hodgkin's lymphoma
Bronchus, Chronic bronchitis	Nasal cavity, Allergic rhinitis
Colon, Adenocarcinoma	Ovary, Carcinoma
Colon, Ulcerative colitis	Pancreas, Carcinoma
Heart, Congestive heart failure	Prostate, Benign hyperplasia
Heart, Myocardial infarct	Prostate, Carcinoma
Heart, Diabetes	Skin, Melanoma
Kidney, Diabetic nephropathy	Small intestine, Crohn's disease
Kidney, Hypertension	Synovium, Rheumatoid arthritis
Lung, Asthma

Because each antibody targets a different epitope on the same protein target, the authenticity of individual cell staining observations can be supported through concordance analysis. Read more about how LifeSpan’s antibodies are selected characterized [LifeSpan’s antibody generation and characterization protocol].

Sign in for access to the DrugTarget^TM database. Contained within the DrugTarget^TM database are three sample gene reports.