Steven A. Fuller

Immunoblotting and immunodetection.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Enzyme‐Linked Immunosorbent Assays (ELISA)

This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell‐surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid‐phase reactants. In the first four protocols, solid‐phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid‐phase reactants are cell‐associated molecules. In all protocols, the solid‐phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. Support protocols are provided for optimizing the different ELISAs and preparing lysates for use as test antigen from bacterial cultures containing expressed protein.

Cloning of Hybridoma Cell Lines by Limiting Dilution

Cloning by limiting dilution is a method based on the Poisson distribution. Dilution of cells to an appropriate number per well can maximize the proportion of wells that contain one single clone. Two or more cloning procedures are carried out until >90% of the wells containing single clones are positive for antibody production.

Immunization of Mice

In the protocols in this unit, antigen is prepared for injection either by emulsifying an antigen solution with Freunds adjuvant or by homogenizing a polyacrylamide gel slice containing the protein antigen. Mice are immunized at 2- to 3-week intervals. Test bleeds are collected 7 days after each booster immunization to monitor serum antibody levels. Mice are chosen for hybridoma fusions when a sufficient antibody titer is reached.

Immunoblotting and Immunodetection

This unit provides protocols for immunoblotting, which is used to identify specific protein sequences separated by electrophoresis and transferred to an appropriate membrane and recognized by a polyclonal or monoclonal antibody. After the proteins are separated on a gel, they are transferred to a membrane by electroblotting or a semidry transfer system. Proteins on the membrane can be visualized with the reversible stain Ponceau S to assess the completeness of transfer. Then the blot is analyzed with antibodies. The primary antibody is specific for the protein(s) of interest; the secondary antibody (an anti‐Ig) is conjugated to horseradish peroxidase or alkaline phosphatase and detected colorimetrically or by chemiluminescence. The membrane can be stripped and reused for other probes.

Fusion of myeloma cells with immune spleen cells.

Liquid nitrogen storage is the method of choice for long‐term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.

UNIT 6.2 Immunoblotting and Immunodetection

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS‐PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody‐antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti‐IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. Curr. Protoc. Cell Biol. 52:6.2.1‐6.2.28.

Conjugation of Enzymes to Antibodies

Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen‐specific monoclonal or polyclonal antibody in which neither the antigen‐combining site of the antibody nor the active site of the enzyme is functionally altered. This unit describes procedures for cross‐linking HRPO, urease or alkaline phosphatase to immunoaffinity‐purified monoclonal or polyclonal antibodies (IgG).

Purification of Monoclonal Antibodies

The uses of monoclonal antibodies as enzyme conjugates and as immunoaffinity reagents require their purification from the crude ascites fluid. This unit provides protocols for purification using protein A-Sepharose chromatography and affinity chromatography, which are superior to ammonium sulfate precipitation, gel filtration, or ion-exchange chromatography for the preparation of contaminant-free antibody.

Design and production of bispecific monoclonal antibodies by hybrid hybridomas for use in immunoassay.

Publisher Summary This chapter describes methods utilized or developed to prepare a panel of fusion partners that simplifies the production of bispecific monoclonal antibodies (bsMAbs), the preparation of hybrid hybridomas, and the production and use of bsMAbs. In conventional use, monoclonal antibodies (MAbs) are covalently coupled to various reagents (e.g., enzymes, fluorochromes, radioisotopes, dyes, and toxins). Enzyme immunoassays (EIA) utilize MAb cross-linked to any of several marker enzymes. Such chemical modification of both antibody and enzyme has several limitations: (1) an antibody–enzyme mixture that includes both active and inactive species in varying proportions, (2) a preparation that contains both antibody and enzyme coupled to contaminants as well as to each other, (3) loss of antibody function, and (4) antibody–enzyme conjugates that are often less stable than the unmodified components.