Here are Some Useful Terms

A variety of chromatographic methods are performed. These may include size exclusion chromatography for native protein size, ion exchange, hydrophobic, reverse-phase based upon solubility separation, dye-binding and other chromatographic methods to characterize proteins.
A bioassay measures a biological response of a cell or organ to a drug, agonist or antagonist. The types of responses include growth/inhibition/death/apoptosis, induction of gene expression (measured by qPCR)/protein production (ELISA,Western, flow cytometry)/morphological changes, etc.
A biomarker is a detectable response in cells or in the body to an intervention. It can indicate a particular disease state. Biomarkers can indicate deleterious side effects of drugs, risk of progression of a disease, or susceptibility of the disease to a given treatment.
A bioassay measures a biological response of a cell or organ to a drug, agonist or antagonist. The types of responses include growth/inhibition/death/apoptosis, induction of gene expression (measured by qPCR)/protein production (ELISA,Western, flow cytometry)/morphological changes, etc.
B lymphocytes, or B cells, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system by secreting antibodies.
Killing of a target cell by immune cells when directed by a specific antibody to the target. A positive aspect if the antibody is designed to kill cancer cells. A negative aspect if the antibody is designed for other functions. Measured by radioactive 51Cr release from the target or by other methods.
Killing of a target cell by immune cells when directed by a specific antibody to the target. A positive aspect if the antibody is designed to kill cancer cells. A negative aspect if the antibody is designed for other functions. Measured by radioactive 51Cr release from the target or by other methods.
Flow cytometry measures physical and chemical characteristics of a population of cells or particles.[1][2][3][4] A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. The sample is focused to ideally flow one cell at a time through a laser beam and the light scattered is characteristic to the cells and their components. Cells are often labeled with fluorescent markers so that light is first absorbed and then emitted in a band of wavelengths. Tens of thousands of cells can be quickly examined and the data gathered are processed by a computer.
Drug action can be followed in a bioassay in which a cell has to respond to a signal (as contrasted to simply binding to a cell receptor). Bioassays may be developed to detect the drug being transported into intracellular compartments or may measure intracellular signaling events such as receptor phosphorylation, calcium flux, or gene activation (Reporter Assays, PCR). Bioassays could measure metabolic effects and other biological events on cell growth or death. Additionally, bioassays could detect induction of new protein synthesis and protein secretion and cell component rearrangements.
Gene expression, the activation of a specific gene to produce a messenger RNA that directs the synthesis of protein. Other gene expressions include RNA molecules from non-protein coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes.
Granulocytes are a category of white blood cells characterized by the presence of granules in their cytoplasm. They are also called polymorphonuclear leukocytes or polymorphonuclear neutrophils (PMN, PML, or PMNL) because of the varying shapes of the nucleus, which is usually lobed into three segments. They are part of the innate immune system which comprises an immediate detense against pathogens, as contrasted to lymphocytes which take days to proliferate and become effector cells.
To screen a library of thousands of compounds, a high throughput screening assay will facilitate the identification of potential “hits” in less time than a typical assay. A high throughput assay typically is not complicated, must be very reproducible and have a high signal-to-noise ratio in order to minimize false positives. The method could be a binding assay to cells or to molecular target using ELISA, a bioassay or using a subcellular fraction, or an enzyme activity assay, Hits are then validated by more specific activity assays.
High Performance Liquid Chromatography (HPLC) is one of the most widely used analytical techniques. It utilizes a liquid mobile phase to separate the components of a mixture by forcing the components (analytes) dissolved in solvent to flow through a chromatographic column under high pressure. The mixture is resolved into its components based on the degree of interaction between the solute components and the stationary phase, defined as the immobile packing material in the column. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. The various components in the mixture pass through the column at different rates due to differences in their distribution between the mobile liquid phase and the stationary phase. Each protein has a characteristic peak under given chromatographic conditions and should have a reasonable retention time and be well separated from other extraneous peaks. Typically, the protein is detected using ultraviolet spectrophotometry.
Immunization of the appropriate specie with antigen emulsified with adjuvant occurs on days 1, 14, and 28. Sera are tested for antibody titer in an ELISA assay (or with the investigator’s screening method) and if titers are high, hyperimmunization is performed and spleen cells are isolated and fused to myeloma cells with polyethylene glycol (PEG). Hybridoma cells are selected with HAT medium that kills non-fused myeloma and spleen cells. Hybridoma cells are subcultured in microwells and supernatants are screened for IgG secretion in the investigator’s antigen specific system. Cells from positive wells are cloned. A solid phase ELISA assay or FACS screening can be used to identify positive wells. The positive cultures are cloned and the culture supernatants rescreened. Frozen stocks are produced as well as the conditioned media that contains the monoclonal antibody.
Immune Checkpoint Inhibitor (ICI) refers to therapy that targets immune checkpoints, key regulators of the immune system which tumors use to protect themselves from attack by the immune system. The initial approved drugs are antibodies that block these regulators.
Cellular immunoassays include the activities of B and T lymphocytes and the subsets of granulocytes and monocytes/macrophages.
The presence and the cellular localization of macromolecules can be determined by immunocytochemistry, in which cells are fixed on a microscope slide, and a molecule is stained by a specific labeling reagent and detected by fluorescence microscopy. Molecules external to the cell membrane are visualized directly whereas internal molecules require permeabilized membranes.
The IgM, IgG subsets or other classes of antibody are determined by immunoassay methods. The kappa or lambda light chain class is similarly determined.
This technique detects specific markers on subsets of cells, e.g. tumor cells or subsets of T lymphocytes. The method may be performed on single cell samples by flow cytometry or immunofluorescent microscopy, or on tissue sections by immunohistochemistry.
In situ hybridization localizes the expression of a particular mRNA among different cells in a tissue or cell preparation. A cell preparation is fixed on a slide and the RNA contents probed with radiolabeled or fluorescent probe.
Ion exchange chromatography separates proteins or peptides based on charge characteristics. The net surface charge of a protein or peptide determines its adsorption to oppositely charged groups immobilized on the ion-exchange medium. Proteins are multivalent anions or cations, and the charge of a protein depends on the pH of the environment. When the pH is greater than the isoelectric point (pI) of the protein (number of positive charges equals the number of negative charges), the protein will have a net negative charge and will bind to an cation (+) resin. When the pH is less than the isoelectric point, the protein will have a net positive charge and will bind to an anion (-) resin. Once the sample is bound to the medium, unbound components are washed away and bound samples selectively eluted and collected.
Cells of the immune system are commonly purified from blood, spleen or lymph nodes. Separate cell populations (lymphocytes, granulocytes and monocyte / macrophages, erythrocytes, and cancer cells) are usually prepared by density gradient centrifugation through Ficoll-Hypaque or Percoll solutions. Separation is based on the buoyant density of each cell subpopulation at the given osmolality of the solution. Monocytes and neutrophils are also purified by selective adherence. If known subpopulations are to be isolated, for example CD4+ or CD8+ T cells, fluorescence activated cell sorting (FACS) will be employed or magnetic beads coated with specific anti-CD4 or anti-CD8 monoclonal antibody are used. The beads are mixed with peripheral blood leukocytes and only CD4+ or CD8+ cells will bind to the beads, which are then separated out from the non-specific cells with a magnet. Another method depends on killing the undesired populations with specific antibodies and complement. In some cases, a noncytotoxic antibody or other inhibitor can block the activity of a cell subtype. Characterization of cell types and subpopulations can be performed using markers such as specific enzymes, cell surface proteins detected by antibody binding, cell size or morphological identification.
A DNA library is screened to determine the presence of a gene and to clone out either a full-length or smaller version of the gene. There are many strategies for cloning from a library. Typically, the library is introduced into bacteria, the bacteria are plated under cloning conditions, replicate plates of the bacterial colonies are made, and the clones are probed for the existence of the desired gene either by hybridization or through the expression of the specific protein. For a positive clone, the DNA is extracted from original plate, recloned and purified.
Macrophages (Greek: big eaters, from Greek (makrós) = large and (phageín) = to eat) are a type of white blood cell, of the immune system, that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the type of proteins specific to healthy body
MHC proteins, found on cell surfaces bind foreign molecules (antigen), from pathogens and display them on the cell surface so that T-cells can recognize the antigen and illicit an immune response by the acquired immune system. They are also important to determine histocompatibility between people for transplantation, blood infusion.
Drug effect on metabolism is measured by radioactive precursor uptake, thymidine, uridine (or uracil for bacteria), and amino acid, into DNA, RNA and proteins. Carbohydrate or lipid synthesis is similarly measured using suitable precursors. Turnover of nucleic acid or protein or the degradation of specific cell components, is measured by prelabeling (or pulse labeling) followed by a purification step and quantitation of remaining label or sometimes by measurement of chemical amounts of the component. Energy source metabolism is also analyzed for optimal cell growth.
microRNA (miRNA) are small RNAs that direct enzyme complexes to degrade messenger RNA molecules and thus decrease their activity by preventing translation.
Mixed Lymphocyte Response (MLR) a test used by to show the safety of a drug or implantable material. It is commonly used as part of the FDA clearance process. Blood lymphocytes of one or two donors are cultured. If there is a detectable mismatch, the lymhocytes will proliferate and demonstrate activation markers and new protein cytokine synthesis.
Monocytes are a type of leukocyte, or white blood cell. They are the largest type of leukocyte and can differentiate into macrophages and myeloid lineage dendritic cells. As a part of the vertebrate innate immune system monocytes also influence the process of adaptive immunity.
Multiplex is a type of immunoassay that uses a mixture of uniquely labeled beads to simultaneously measure multiple analytes in a single sample. A multiplex assay is a derivative of an ELISA using beads for binding the capture antibody. Other examples are quantitation of multiple RNA species.
Natural killer (NK) cells are an essential defense in the early stage of the immune response to pathogens. NK cells are active in naïve individuals and their numbers can be enhanced in certain circumstances. The NK assay typically uses a 51Cr-labeled tumor target and is similar to the CTL assay described above.
A drug may elicit an immune response in recipients. The ELISA is the typical method for detecting antibodies that bind to a drug. The Neutralizing Antibody Assay (NAb) measures whether the antibody blocks the activity of the drug. The assay usually is a bioassay in which the drug induces a biological response in a cell and patient serum antibody is tested for inhibition that response.
Neutrophils are the most abundant type of granulocytes and the most abundant (40% to 70%) type of white blood cells in most mammals. They form an essential part of the innate immune system.
Natural Killer (NK) cells are a type of lymphocyte (a white blood cell) and a component of innate immune system. NK cells play a major role in the host-rejection of both tumours and virally infected cells.
Northern blot analysis involves size separation of the RNA species by electrophoresis followed by identification with labeled gene-specific oligonucleotides or DNA probes. The size and abundance of the desired RNA is determined. The predominant rRNA in a total RNA preparation can also be detected by UV absorbance as a measure of intactness.

PCR

PCR is a very sensitive method for detecting specific DNA and RNA segments. It is used to determine whether a gene is present in the genome or to detect the level of gene expression in the case of drug action or genetic manipulation.
Peptides or haptens can be conjugated with various carrier proteins for use as immunogens in animals such as mice, rabbits and goats. Conjugates are also useful for affinity purification of antibodies or other binding proteins. Keyhole limpet haemocyanin (KLH) is routinely used as a carrier protein, with conjugation of the peptide to the carrier protein typically through an N-terminal or C-terminal amino acid. Other carrier proteins (e.g., ovalbumin, tetanus toxoid, diphtheria toxoid, tuberculin PPD) and conjugation procedures (e.g. glutaraldehyde, bis-diazotised tolidine (BDT), carbodiimide) are available upon request.
Peptides or haptens can also be conjugated to various fluorochromes or to biotin, through covalent attachment to lysines, thiols or carbohydrates and subsequently detected with either avidin or streptavidin. Peptides can also be radiolabeled. These conjugates can be used for ELISA or RIA, or for analysis of receptor binding and cellular uptake.
Light microscopy shows the general state of cells, and combined with trypan blue exclusion, the percent of viable cells. Small, optically dense cells indicate necrosis, while bloated “blasting” cells with blebs indicate apoptosis. Phase microscopy views cells in indirect light; the reflected light shows more detail, particularly intracellular structures. Fluorescence microscopy detects individual components in cells, after labeling with selective dyes or specific antibodies, and can distinguish cell surface from intracellular labeling. Microscopic observation of cell cultures is an integral tool for tissue culture, as it reveals the culture health during the maintenance, expansion and experimentation phases of the study. It contributes to the “art” of cell biology.
Polyclonal Antibodies are isolated from serum obtained from a variety of species. Serum is prepared from test bleeds and assayed for high titer antibody, followed by production bleeds after continued boosting.
A potency assay is a GMP lot release assay that measures the activity of the drug in units per mg. Typically the assay is a bioassay in which increased concentrations of drug induce a sigmoid shaped response curve of a biological event. The EC50 determination defines the
Protein concentration is determined by absorbance at 280nm (aromatic amino acids) or 205nm (peptide bond), by the Bradford assay or by amino acid analysis.
Enzymes, antibodies and other proteins can be purified from natural sources such as cells, tissues or plants, or produced recombinantly in bacteria, yeast and mammalian cell culture or in the baculovirus insect cell system. Fermentation lot sizes vary up to 1gm of specific recombinant protein. The desired protein can be purified to homogeneity. Protein identity can be determined by a biological or enzyme activity, immunoassay, and amino acid sequence. Quality control can use a variety of biochemical assays.
Quantitative PCR (qPRC). qPCR can measure the amount of specific RNA that is being regulated by normal gene expression as well as drug influenced gene expression.
The RadioImmunoAssay (RIA) uses radiolabeled molecules to measure very low concentrations of substances. The format is usually a competition between the analyte being tested and a radiolabeled version of the same molecule. The method may use a typical ELISA plate or a precipitation method to separate bound from free analyte.
Use of radioisotopes offer extreme sensitivity for enzyme activity assays and detection of proteins, nucleic acids and other markers. We have licenses for 3H for labeled thymidine uptake into DNA, 32P for various nucleic acid assays, 14C and 35S for amino acid and protein studies, 45Ca for metabolism studies, 51Cr for target killing tests and 125I for radiolabeling many compounds.
Receptor function and quantitation is accomplished by binding its labeled ligand (growth factor, metabolic trigger, cancer drug, etc.) or other specific agents such as antibodies. Nonspecific binding is also measured and corrected specific binding is calculated. Some receptors are internalized and degraded following binding of their ligand and this can be measured. Activation of tyrosine kinase receptors is followed by phosphorylation that can be measured. Membrane preparations can be used for direct binding assays for quantitation of ligands
Gene activation or gene expression can be conveniently monitored by a DNA construct that links the regulatory components of the gene to a reporter gene, such as for a fluorescent protein or beta galactosidase. Cells can be transfected with reporter genes that are activated when certain pathways are triggered. Pathway induction is quantitated by the reporter gene, such as the appearance of fluorescence or of an enzyme activity. These surrogate methods may be much more sensitive and rapid than detection of the primary gene response.
Multi-gram quantities of antibodies or proteins can be produced by suspension culture (125 mL to 15 Liter bioreactor flasks) in serum-free medium. Antibodies can be further purified by various methods.
Signal transduction is the process by which a chemical or physical signal is transmitted through a cell as a series of molecular events, most commonly protein phosphorylation which results in a cellular response. Protein receptors detect stimuli from ligand agonists which trigger the process. At the molecular level, such responses include changes in the transcription or translation of genes, and post-translational and conformational changes in proteins, as well as changes in their location.
Small interfering RNA (siRNA) is a class of double-stranded RNA molecules, 20-25 base pairs in length. They interfere with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. It “silences” the specific gene expression.
The limit of solubility and adjustment of salt, pH and formulation to increase solubility are performed. Solubility in a variety of detergents are investigated to retain protein or enzyme activity and conformation when developing isolation methods from cell membranes.
There are a variety of strategies to stabilize proteins and enzymes. Storage of the proteins is investigated including lyophilization, flash freezing, normal freezing methods, and storage below 0oC with glycerol or DMSO like compounds. If kept at 2-8oC, the types of potential antibacterial reagents is determined. If kept at 2-8oC without antibacterial reagents, the method of generating a sterile protein solution without adsorption loses onto a filter is identified. The addition of stabilizing reagents is methodically studied so that bioactivity and physical conformation is not affected.
Cells can be fractionated when purification of a specific subcellular component, or the activity of the component, or investigation of drug localization is required. Cell fractions include plasma membrane, nuclei, mitochondria, cytoplasm, nucleoplasm, microsomes. Purity of each fraction is assessed with enzymatic markers.
Cell surface markers define cell subsets and state of activation. Most markers are receptors for agonists or regulators of cell function. The typical analytical method is flow cytometry.
Recombinant proteins are expressed in bacteria, yeast, mammalian or in the baculovirus system. The gene of interest is transfected into the appropriate host together with regulatory elements that allow very high levels of production. Protein production may be intracellular, or may accumulate in the bacterial periplasmic space, or be secreted into the medium of cells. The scale may be a few mL for analytical purposes, or in batches of up to 20 L in bacterial or yeast fermentation, or up to 50 L in mammalian suspension, porous bead cultures.
Classical PCR greatly amplifies a specific sequence of DNA for analysis or molecular cloning. qPCR starts with mRNA or total RNA, makes a cDNA complimentary strand using reverse transcriptase, and then amplifies the product. The amount of cDNA, and therefore mRNA, present in a sample can be quantitatively determined based on a standard of known quantity, or qualitatively assessed relative to the level of mRNA in a control sample.
T lymphocytes comprise the cellular component of the adaptive immune system. They include helper cells that initiate immune reactions, and effector cells that mediate killing of target cells. See T cell subsets
T lymphocytes comprise the cellular immunity function. The main categories are grossly CD4+ (T4) helper cells which aid in antibody production by B lymphocytes, and CD8+ (T8) cytotoxic effector cells for eliminating virus-infected cells, tumor cells, etc.. Each main group has many subtypes and differential functions.
The T-helper cell 17 (Th17) lineage is a subset of effector memory T cells. Th17 cells play a critical role in the induction of the tissue inflammation and tissue destruction that are hallmarks of many immune-inflammatory diseases.
Transfection is the process of introducing nucleic acids (genes) into eukaryotic cells. The result may be to overexpress certain proteins, to correct a genetic defect, or to create a reporter cell that conveniently fluoresces when triggered by an agonist.
Purified, full-length RNA is required for cDNA cloning. It is also used in some methods for analyzing gene expression and for microarrays utilizing cDNA. RNA is prepared from bacteria, tissue culture cells, tissues and plants. Preparations can be purified messenger (mRNA) or total RNA that includes mRNA, ribosomal (rRNA) and transfer RNA (tRNA). The scale is from 1 microgram to1 milligram.
The Western blot identifies specific protein antigens and their approximate size. Proteins are separated by polyacrylamide gel electrophoresis (PAGE) after denaturation with sodium dodecyl sulfate (SDS) which makes proteins more linear and migrate in inverse proportion to their molecular weight. A unique protein band is detected by “blotting” or electrophoretically transferring the protein onto nylon or nitrocellulose support. Detection of the protein band results when the support is incubated with a specific antibody that is conjugated with a radiolabel, enzyme or other method. The dot blot is a simpler method involving binding a small amount (10 microliters) of a protein mixture onto a solid support and detecting it as described above. Both methods can be quantitative by use of concurrently run standards.
Complement Dependent Cytotoxicity (CDC) is an effector function of IgG and IgM antibodies. When they are bound to surface antigen of a tumor or other cell, complement protein in blood are triggered, resulting in target cell lysis. This is a desired characteristic for a cancer therapeutic, and undesirable for other antibody drug.
Specifically activated lymphocytes synthesize and secrete a number of distinctive cytokines. These are quantitated by various ELISA methods. Alternatively, induced cytokines are detected by fluorescence activated flow cytometry (FACS) using fluorescent antibodies that bind to the membrane surface or enter permeabilized cells. Activated cells also express new cell surface antigens where the number of cells is quantitated by immunofluorescent microscopy, flow cytometry, or ELISA. Unique cell surface receptors that distinguish cell populations are detected by similar immunochemical methods or by the binding of their specific labeled ligand.
Cells secrete protein messengers called cytokines. These are measured by ELISA, by flow cytometry of the producing cells, or in a functional assay.
A key activity of cellular immunity in rejection of transplants, reactions to pathogens such as viruses and tumors, is the development of T lymphocytes that specifically kill target cells. These activated cells develop during in vivo exposure or by in vitro sensitization. The CTL assay consists of increasing number of sensitized lymphocytes cultured with a fixed number of tumor or other target cells that have been prelabeled with 51Cr. To prelabel the target cells, the cells are incubated with the radiolabel 51Cr. Susequently 51Cr is taken up and reversibly binds to cytosolic proteins. When these target cells are incubated with sensitized lymphocytes, the target cells are killed and the 51Cr is released into the supernatant.
Protein, peptide or coding DNA sequences are searched against databases to determine uniqueness, related families of proteins, and to suggest characteristic properties of a protein.
Any given protein (antibody) can be separated from other proteins based on its unique physical and chemical properties. Proteins in solution form hydrogen bonds with water through their charged and polar side chain groups. If the protein solvent interaction is prevented, proteins can interact with each other and form aggregates that precipitate out of solution. As the concentration of salt is increased in a solution, the amount of water available to interact with protein is reduced, leading to interaction between hydrophobic groups on different proteins and the formation of a precipitate. Various factors, including the molecular weight of the protein, pH of the solution and temperature can affect the concentration at which a particular protein will precipitate out of solution. Ammonium sulfate is the salt most commonly used to precipitate proteins from solution. Organic, water miscible solvents (ethanol, methanol, acetone) can also be used to differentially precipitate out proteins.
Purified DNA is required to prepare DNA libraries or microarrays, to make probes, and to transfect genes. DNA is prepared from bacteria, yeast, tissue culture cells, tissues, or plants. Preparations can be genomic DNA or plasmids. The scale is from 1 microgram to 1 milligram.
Restriction endonucleases recognize and cut DNA at unique nucleotide sequences. Delineation of these cleavage sites provides a map of the DNA, produces characteristic sizes of the fragments as detected on an agarose gel, and allows for the preparation of pieces of DNA to be used for making new vector or plasmid constructs.
To unequivocally identify a gene or cDNA, the DNA is submitted for sequencing. Using overlapping portions of the DNA, a contiguous sequence is generated which can be used for data mining.
Subcloning is used to amplify a desired DNA molecule. After a piece of DNA or a gene is obtained, by synthetic synthesis, restriction digestion, joining to another DNA by a ligase, etc., it is introduced into competent bacteria and transformants expressing the new gene are identified. The bacteria are cloned on selection plates whereby only those cells that contain the desired DNA survive and propagate into colonies. The colonies are “picked” and expanded in liquid cultures.
Specific RNA species in an unfractionated preparation can be measured by immobilizing a sample in a spot (Dot Blot) or in a manifold slot (Slot Blot). Detection is by a labeled DNA probe that hybridizes to the immobilized RNA. For the Dot Blot, quantitation is usually visual whereas the Slot Blot format is more easily quantitated by scanning with a densitometer.
Drugs may be presented to biological systems by themselves, as prodrugs that have to be metabolized to a form more readily taken up or in liposomes that facilitate transport across lipid membranes. Drugs may also be formulated in degradable polymers or other slow-release systems or attached to carriers to facilitate transport such as nanoparticles, to decrease clearance or to maintain circulating concentrations. Drug capture by a cell surface receptor is measured by localization (subcellular fractionation), or by its action of triggering a biological reaction (cell signaling, reporter gene assays, proliferation, cell death, metabolic assays). The concentration of drug in its initial formulation, and of free drug, internalized drug, and subsequently metabolized or degraded drug can be measured.
Purity is assessed by SDS-PAGE and/or nondenaturing electrophoresis for molecular weight and disulfide polymerization. Isoelectric focusing can be performed for identifying the isoelectric point (pI) of a protein. To determine homogeneous pure proteins, N-terminal amino acid sequencing is used. Other methods can be developed upon request. Electrophoresis describes the migration of charged particles under the influence of an electric field. It can be used to evaluate protein purity and provide an estimation of characteristics such as isoelectric point, charge, and subunit composition. Gel electrophoresis is the technique in which molecules are forced across a span of gel by an electrical current. Proteins, peptides, amino acids, nucleotides etc. contain groups that can ionize and at any given pH, exist in solution as electrically charged species either as cations (+) or anions (-). Separation of large (macro) molecules depends upon two forces: charge and mass. During electrophoresis, the rate of migration in the electric field depends on the strength of the field, relative hydrophobicity of the samples, size and shape of the molecules, and on the ionic strength and temperature of the buffer in which the molecules are moving. The most common one-dimensional gel methods use sodium dodecyl sulfate (SDS), which binds to proteins in a uniform amount per microgram of protein. This results in a uniform charge density per unit mass, providing a separation based on the mass of the polypeptide chain. Proteins can be separated for further purification by isoelectric focusing (IEF) in slab gels in which the movement of proteins through pores in a polyacrylamide gel matrix is controlled by a pH gradient created by soluble ampholytes.
Enzyme-Linked ImmunoSorbant Assay is a quantitative and sensitive assay which quantifies substances such as antibodies, hormones, proteins and peptides even in a complex matrix such as serum or cell homogenate. Applications include RadioImmuneAssay (RIA) for small molecules and ELISPOT, which detects signals (e.g. cytokine) from a single cell. Our scientists have decades of experience developing these methods, even at pg/mL levels in a complicated matrix such as serum, with detection levels of 1 part per 10 million.
ELISpot or enzyme-linked immunosorbent spot, allows for the detection of cells that secrete cytokines or antibodies. ELISpot is able to detect a single cell that secretes a protein of interest making it one of the most sensitive cellular assays. Cytokine-specific antibodies are placed onto an ELISpot plate. Cells of interest are then added. Cells are activated and produce cytokine which binds to the antibodies, which are detected by an enzyme-substrate method. A colored product produces a spot that is counted through a microscope
The Limulus Amebocyte Lymph (LAL) assay is used to quantitate pyrogen (endotoxin). Methods are used to remove endotoxin for biological assays.
Marin Biologic is experienced in many types of assays, including those employing proteases, dehydrogenases, oxidases, phosphorylase, nucleic acid polymerase and modifying enzymes, and glycolytic enzyme assays. Colorimetric, fluorescent and radiometric formats are used. Substrate specificity, ED50, ID50, substrate and inhibitor kinetics, temperature dependence and sensitivity are factors in selecting an assay. Assays for glucose, glutamine, lactic acid, endotoxin, etc. are also performed.
Enzymes and proteins are tested for stability to temperature, pH, chemical denaturation, and proteolytic degradation. Methods for stabilizing enzymes and proteins during storage are developed.
Epitope mapping is the identification of the specific regions of a protein recognized by the antibody binding site. The protein is clipped into fragments, or fragments are expressed recombinantly, and tested for binding by antibody. This narrows down the protein region of antibody binding. A series of synthetic peptides containing overlapping amino acid sequences based on the protein structure are also constructed and the exact epitope recognized by the antibody can be determined by analyzing which peptides are bound by the antibody. This would be a linear determinant epitope. Some epitopes depend on a tertiary conformation structure and antibody binding may only occur with large protein fragments
Purification of a protein is accomplished by the initial binding of the protein to an immunogen, in the case of an antibody, or to a substrate in the case of an enzyme. Other affinity purifications could be an immobilized DNA for a DNA-binding protein, immobilized ligand for a receptor-ligand pair or an immobilized ligand that binds a tag on a recombinant protein. Affinity columns can be made by covalently crosslinking the ligand to the support gel, such as Sepharose. The bound protein is separated from contaminating proteins by washing the column thus eluting contaminants, and subsequently eluting the protein of interest by acid or chaotropic reagents followed by recovery of activity. Typically, good separations of the protein from contaminants are achieved so that only one or two subsequent purification methods will resolve the protein to nearly homogeneity.
The N-terminal amino acid sequence of a purified protein is determined to confirm its identity, the integrity of the N-terminus, and to verify that the preparation is homogeneously pure. Total amino acid analysis is performed to confirm the identity and full length of the protein and its absorbance at 280 nm.
Antibodies are conjugated to provide reagents for analysis. This includes addition of radiolabel, typically 125I on tyrosine resides, enzymes, fluorochromes, or biotin. Conjugates are used for ELISA, Western blot or immunohistochemistry studies. Biotinylated antibodies are detected by labeled streptavidin to which it binds very strongly.
Antibodies are isolated from serum obtained from a variety of species. Serum is prepared from test bleeds and assayed for high titer antibody, followed by production bleeds after continued boosting. Antibody purification may include an ammonium sulfate precipitation and affinity purification methods. It may also include classical methods such as ionic separations, ultrafiltration, etc.
The strength of antibody binding to its ligand is assessed by radioimmune assay (RIA), ELISA, or binding in a column support format. Dissociation is performed by increasing denaturating conditions, or by competition with a related ligand. The dissociation constant, Kd, is determined by a Scatchard plot.
A protein is characterized by binding to its receptor or ligand. The protein is bound to a solid support (ELISA plate, beads, column), incubated with labeled ligand, and the amount of bound ligand is determined. Equilibrium dialysis can be performed for precise Kd affinity values when applicable. Alternatively, the receptor may be on a cell surface. The affinity of the interaction, measured by the dissociation constant, Kd, is determined. These analyses may involve a UV spectophotometric, colorimetric, fluorescence or radioactivity method of detection.
Glycoproteins are specifically stained after SDS-PAGE fractionation and differentiated from unglycosylated proteins. Deglycosylation can be performed as well.
A library is an amplified collection of DNA representing the total DNA of an organism (genomic library) or of DNA complementary to a preparation of mRNA (cDNA library). Either library can be probed to pull out a specific gene of interest. cDNA libraries are used to clone the expressed version of a gene comprised of the exons. Genomic libraries contain both the exons, introns and regulatory portions of the gene. Bacteriophage lambda and cosmid vectors are used in library construction.
Each cell line must be matched to a particular growth medium. As a generalization, ectodermal cells such as in the fibroblast lineage are adherent and prefer one subset of media, while blood cell types are nonadherent and prefer different formulations. Formulations usually include glucose as an energy source, vitamins, amino acids and 5 – 20% calf serum. Alternative energy sources are sometimes used. Small scale growth and maintenance in culture (1 mL to 100 mL) is carried out in tissue culture grade plastics, while scale-up utilizes roller bottles, porous bead supports or a hollow fiber bioreactor, or stir cells. Nonadherent cells are also economically grown up to 40 L in stir cell suspension culture. Some adherent cell types can be adapted to nonadherent growth resulting in a more efficient production method. Adaptation to serum-free conditions allows convenient purification of a secreted protein product without the contribution of albumin.
DNA complementary (cDNA) to mRNA is prepared for preparing microarrays for screening in different cell sources. For screening, the total RNA is extracted from tissue or cells and is hybridized to the cDNA immobilized onto a solid support. A detection molecule is applied and is visualized.
Purified or unseparated lymphocytes can be activated for proliferation and DNA synthesis can be measured by a variety of techniques: dye method MTT, Alomar blue, CTG, or 3H-thymidine incorporation, or activation by cytokine production, expression of activation antigens, or increase in cell size. Activation is accomplished by incubating cells with nonspecific activators such as Concanavalin A, phytohemagglutinin (PHA), phorbol myristic acetate (PMA), an ionophore, an antibody to T cell receptors, or stimulation with specific antigen to which the cells are sensitized.
Cloning is used to obtain a stable culture of homogeneous cells. Over periods of many months in tissue culture, cells can change properties due to somatic gene mutation, and overgrowth of mutated cells. It may be desired to select a rare cell type or the few stably transfected cells in a transfection pool. Cloning is achieved by diluting a culture so that ½ the wells in a 96 well plate contain one cell and ½ contain no cells. At this low cell concentration, conditioned medium (medium from the same cell type harvested at high concentrations) may be added to enhance growth. When the clone reaches suitable numbers, aliquots are frozen in order to retrieve cells with the same properties at a future date.
Cell signaling for a number of activities is measured by a variety of techniques, such as calcium flux, change in intracellular pH, metabolic assays, proliferation, and gene expression
For most studies, cell growth is measured by a homogeneous, vital dye method in which one of several choices of dye is added to cells in a 96 well plate at the conclusion of the study or at time point intervals, and read directly in a plate reader. The dye is enzymatically changed in healthy cells so that development of color or fluorescence is measured using a different wavelength than the unaltered dye. The effects of adding a growth factor, an inhibitor, or a cytotoxic factor to cells is easily read. This procedure has very few steps, has minimal manipulation of cells, and allows for good reproducibility. Alternatively, uptake of 3H-thymidine is used specifically to assay DNA synthesis, or as a more sensitive assay of cell proliferation for slow growing cells. Cell death occurs by lysis, necrosis, or apoptosis. Lysis is the destruction of the cell surface membrane such as by the action of an antibody and complement that makes holes in the membrane. Necrosis occurs through the action of toxic factors that act within the cell, such as irreversible inhibitors of protein, RNA or DNA synthesis, or mitotic poisons. Apoptosis is a programed cell death used by the body to remove damaged or unwanted cells and occurs during cytotoxic T cell killing and with some cancer chemotherapies. Apoptosis is characterized by early events such as expression of phosphotidylserine on the cell surface and fragmentation of the DNA, followed by loss of membrane integrity and mitochondrial function. The quantitative percentage of dying cells is determined microscopically or by flow cytometry using vital stains or DNA-binding dyes. High throughput measurement of cell death is performed by release of a label from cells prelabeled with a radiotracer, typically 51Cr, or a fluorescent or color marker. Alternatively, the fluorescent or colorimetric dye method described above is used.
Cell lines maintain growth and specialized properties during prolonged or indefinite culture in the laboratory. Primary cell isolates are derived fresh from tissues and will grow and maintain specialized properties for a limited time, about 10 passages. Marin Biologic has experience with explants of liver, breast, ovary, lung, skin, spleen, lymph node and brain. Both cell lines and primary cultures can be stored frozen in liquid nitrogen and then put back into culture. These methods facilitate biological studies that are convenient, reproducible, and cost effective. Cell lines allow studies that may be difficult with whole organs or in vivo. These studies include mechanism of action, radioactive experiments, or system manipulation. Cell lines with desired properties are obtained from repositories such as the American Type Culture Collection (ATCC) or derived by Marin Biologic through selection techniques and/or DNA transfection (see below).

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