large quantities of antibody. blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen—in the serum, which can now be called antiserum. antigens are also injected into chickens for generation of polyclonal antibodies in egg yolk. to obtain antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. the fused cells are called hybridomas, and will continually grow and secrete antibody in culture. single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies. polyclonal and monoclonal antibodies are often purified using protein a/g or antigen-affinity chromatography. in research, purified antibodies are used in many applications. antibodies for research applications can be found directly from antibody suppliers, or through use of a specialist search engine. research antibodies are most commonly used to identify and locate intracellular and extracellular proteins. antibodies are used in flow cytometry to differentiate cell types by the proteins they express; different types of cells express different combinations of cluster of differentiation molecules on their surface, and produce different intracellular and secretable proteins. they are also used in immunoprecipitation to separate proteins and anything bound to them (co-immunoprecipitation) from other molecules in a cell lysate, in western blot analyses to identify proteins separated by electrophoresis, and in immunohistochemistry or immunofluorescence to examine protein expression in tissue sections or to locate proteins within cells with the assistance of a microscope. proteins can also be detected and quantified with antibodies, using elisa and elispot techniques. antibodies used in research are some of the most powerful, yet most problematic reagents with a tremendous number of factors that must be controlled in any experiment including cross reactivity, or the antibody recognizing multiple epitopes and affinity, which can vary widely depending on experimental conditions such as ph, solvent, state of tissue etc. multiple attempts have been made to improve both the way that researchers validate antibodies and ways in which they report on antibodies. researchers using antibodies in their work need to record them correctly in order to allow their research to be reproducible (and therefore tested, and qualified by other researchers). less than half of research antibodies referenced in academic papers can be easily identified. papers published in f1000 in 2014 and 2015 provide researchers with a guide for reporting research antibody use. the rrid paper, is co-published in 4 journals that implemented the rrids standard for research resource citation, which draws data from the antibodyregistry.org as the source of antibody identifiers (see also group at force11). antibody regions can be used to further biomedical research by acting as a guide for drugs to reach their target. several application involve using bacterial plasmids to tag plasmids with the fc region of the antibody such as pfuse-fc plasmid. regulations production and testing traditionally, most antibodies are produced by hybridoma cell lines through immortalization of antibody-producing cells by chemically-induced fusion with myeloma cells. in some cases, additional fusions with other lines have created "triomas" and "quadromas". the manufacturing process should be appropriately described and validated. validation studies should at least include: the demonstration that the process is able to produce in good quality (the process should be validated) the efficiency of the antibody purification (all impurities and virus must be eliminated) the characterization of purified antibody (physicochemical characterization, immunological properties, biological activities, contaminants, ...) determination of the virus clearance studies before clinical trials product safety testing: sterility (bacteria and fungi), in vitro and in vivo testing for adventitious viruses, murine retrovirus testing..., product safety data needed before the initiation of feasibility trials in serious or immediately life-threatening conditions, it serves to evaluate dangerous potential of the product. feasibility testing: these are pilot studies whose objectives include, among others, early characterization of safety and initial proof of concept in a small specific patient population (in vitro or in vivo testing). preclinical studies testing cross-reactivity of antibody: to highlight unwanted interactions (toxicity) of antibodies with previously characterized tissues. this study can be performed in vitro (reactivity of the antibody or immunoconjugate should be determined with a quick-frozen adult tissues) or in vivo (with appropriates animal models). preclinical pharmacology and toxicity testing: preclinical safety testing of antibody is designed to identify possible toxicity in humans, to estimate the likelihood and severity of potential adverse events in humans, and to identify a safe starting dose and dose escalation, when possible. animal toxicity studies: acute toxicity testing, repeat-dose toxicity testing, long-term toxicity testing pharmacokinetics and pharmacodynamics testing: use for determinate clinical dosages, antibody activities, evaluation of the potential clinical effects structure prediction and computational antibody design the importance of antibodies in health care and the biotechnology industry demands knowledge of their structures at high resolution. this information is used for protein engineering, modifying the antigen binding affinity, and identifying an epitope, of a given antibody. x-ray crystallography is one commonly used method for determining antibody structures. however, crystallizing an antibody is often laborious and time-consuming. computational approaches provide a cheaper and faster alternative to crystallography, but their results are more equivocal, since they do not produce empirical structures. online web servers such as web antibody modeling (wam) and prediction of immunoglobulin structure (pigs) enables computational modeling of antibody variable regions. rosetta antibody is a novel antibody fv region structure prediction server, which incorporates sophisticated techniques to minimize cdr loops and optimize the relative orientation of the light and heavy chains, as well as homology models that predict successful docking of antibodies with their unique antigen. however, describing an antibody's binding site using only one single static structure limits the understanding and characterization of the antibody's function and properties. to improve antibody structure prediction and to take the strongly correlated cdr loop and interface movements into account, antibody paratopes should be described as interconverting states in solution with varying probabilities. the ability to describe the antibody through binding affinity to the antigen is supplemented by information on antibody structure and amino acid sequences for the purpose of patent claims. several methods have been presented for computational design of antibodies based on the structural bioinformatics studies of antibody cdrs. there are a variety of methods used to sequence an antibody including edman degradation, cdna, etc.; albeit one of the most common modern uses for peptide/protein identification is liquid chromatography coupled with tandem mass spectrometry (lc-ms/ms). high volume antibody sequencing methods require computational approaches for the data analysis, including de novo sequencing directly from tandem mass spectra and database search methods that use existing protein sequence databases. many versions of shotgun protein sequencing are able to increase the coverage by utilizing cid/hcd/etd fragmentation methods and other techniques, and they have achieved substantial progress in attempt to fully sequence proteins, especially antibodies. other methods have assumed the existence of similar proteins, a known genome sequence, or combined top-down and bottom up approaches. current technologies have the ability to assemble protein sequences with high accuracy by integrating de novo sequencing peptides, intensity, and positional confidence scores from database and homology searches. antibody mimetic antibody mimetics are organic compounds, like antibodies, that can specifically bind antigens. they consist of artificial peptides or proteins, or aptamer-based nucleic acid molecules with a molar mass of about 3 to 20 kda. antibody fragments, such as fab and nanobodies are not considered as antibody mimetics. common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. antibody mimetics have being developed and commercialized as research, diagnostic and therapeutic agents. binding antibody unit bau (binding antibody unit, often as bau/ml) is a measurement unit defined by the who for the comparison of assays detecting the same class of immunoglobulins with the same specificity.