MARIN BIOLOGIC LABORATORIES, INC.
Pre-clinical Contract Laboratory Services
Research, Assay Development, Validation and Testing Services
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IMMUNOLOGY

Cellular Assays Immunoassays Monoclonal Antibody Production
Monoclonal Antibody Manufacture Polyclonal Antibodies
Antibody Purification Antibody Characterization Antibody Derivatives

Cellular Assays

Isolation and Characterization of Cell Populations

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.

Cell Activation

Purified or unseparated lymphocytes can be activated for proliferation and DNA synthesis is measured by ³H-thymidine incorporation. Other measures of activation such as cytokine production, expression of activation antigens, or increase in cell size are utilized. 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.

Mixed Lymphocyte Reaction (MLR)

A common method to assess the cellular immune response is to co-culture lymphocyte populations from two individuals, (e.g. two humans or two mice of different strains) where the T cells recognize the foreign cells from the other strain, activate and proliferate. One population of cells can be restricted to being stimulator cells (e.g. a preparation of tumor cells), by pretreating them with a mitotic poison (mytomycin B), thereby turning the assay into "one-way" MLR.

Cytotoxic T Lymphocyte (CTL) 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. The 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.

Natural Killer (NK) Activity

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.

Cytokine, Activation Antigens and Receptor Analysis

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 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.

Immunoassays

Enzyme-Linked ImmunoSorbent Assay (ELISA)

ELISA assays are very sensitive, precise and quantitative. Some assays such as fluorescence and time-resolve fluoresence are as sensitive as radiometric assays. They can be used to measure an antigen or an antibody, or generally, any macromolecule that binds another molecule or cell. A typical format is to coat a protein on the bottom of a plastic 96 well plate, block remaining potential protein binding sites by incubation with bovine albumin, casein, or other blocking agents, add the test sample which can be in a crude mixture such as serum, wash out any material that does not bind to the first protein, and detect the bound molecule by an enzyme-conjugated specific antibody. Detection is amplified many-fold using an enzyme-linked detection system, because the substrate is continuously turned over and the product is measured. Marin Biologic uses colorimetric, fluorescent, time-resolved, and homogenous (requiring no washing step) ELISA formats.

High Throughput Screening Assays (HTS)

To screen a library of thousands of compounds, a high throughput screening assay will facilitate the number of "hits" in less time than a typical assay. Antibody based immunoassays are often used as the first screen before using cell based high throughput screening assays. 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 follow-up research on the "hits" is time intensive, thus the number of "hits" should enable identification of only active compounds. Additionally, stabilizing the assay components so that production of the completed assay for use in the screens is an objective when developing the initial configuration of the assay.

RadioImmune Assay (RIA)

The RIA uses unmodified antigen (analyte) binding to antibody in competition with radiolabeled analyte. At the end of 1-2 hours of incubation, the fraction of labeled ligand that is bound to antibody is separated from the unbound, free labeled ligand by a precipitation method. The response of the test sample is then compared to a standard ligand curve to determine the concentration of ligand present in the sample.

Western Blot and Dot Blot

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.

Monoclonal Antibody Production

Hybridoma Production of Monoclonal Antibodies

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 Antibody Screening

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.

Clone Selection

The cells from the best five cell cultures producing antibody, will be cloned by limited dilution in order to isolate a monoclonal line. The clones will be tested and the best clones selected for expansion. Hybridomas continue to "throw-out" genes as they are kept in culture (immunoglobulin genes are lost early since they are not needed for survival) and may need to be recloned to find the best producer after 4-6 months in continuous culture.

Monoclonal Antibody Manufacture

Scale-up Production

Multi-gram quantities of antibodies can be produced by ascites production, suspension culture (125 mL to 15 Liter bioreactor flasks) or hollow fiber bioreactor cultures in serum-free medium. Antibodies can be further purified by various methods (see below).

Polyclonal Antibodies

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

Antibodies are purfied using a variety of techniques (see Biochemistry). The most specific method is affinity chromatography (see below). However, if the amount of ligand or antigen is limited, then other methods are used. One method would be differential precipitation using ammonium sulfate, to separate the antibody from albumin and other proteins found in the culture medium or serum. Ion exchange chromatography and HPLC are often used for further purification. Purity is frequently assessed by SDS electrophoresis where the preparation is overloaded on a polyacryamide gel in order to visualize any contaminating proteins.

Affinity Chromatography

In this highly specific technique, antibodies are purified based on high-affinity binding to their ligand. The immunizing protein, antigenic peptide epitope, enzyme etc. is coupled to a column matrix, typically a form of Sepharose, and the corresponding antibody that recognizes the ligand specifically binds to the column and can be separated out from other proteins present in the sample. The antibody is eluted by acid or chaotropic reagents followed by recovery of activity. Due to the high specificity of receptor-ligand interactions, this method yields nearly pure protein with a minimal amount of purification steps.

Antibody Characterization

Immunoglobulin Class

The IgM, IgG subsets or other classes of antibody are determined by immunoassay methods. The kappa or lambda light chain class is similarly determined.

Binding Affinity Determination

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, K_d, is determined by a Scatchard plot. (see Biochemistry)

Epitope Mapping

Epitope mapping is the identification of the specific peptide sequence and its resulting conformational (folded three dimensional) structure recognized by the antibody binding site, specifically, the complementarity determining region (CDR) composed of three hypervariable regions of the Fab portion of the antibody. For large protein antigens, 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.

Antigen Conjugation

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.

Antibody Derivatives

Antibody Derivatives

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.