John G.R. Hurrell

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.

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).

Biosensors in Diagnostics: Potential and Pitfalls

This chapter presents biosensors in diagnostics. The healthcare industry in the United States has been undergoing significant change over the past three years that has provided both, opportunity and barriers to the application of biosensors in diagnostics. Productivity and cost containment pressures have made diagnostic laboratories particularly sensitive to the value of new technology. Capitation contracts to managed care organizations have made many laboratories walk a very thin line between profit and loss. The advent of broader testing and screening for genetic based disorders provides another opportunity for biosensor technology. The discovery of the multiple mutations involved with many disorders, such as cystic fibrosis and the multigenic basis of cancer and other diseases, makes conventional testing difficult and expensive. Biosensor technology may provide the answer to rapid and inexpensive genetic testing in the future. Biosensors have some unique attributes that make them particularly suited for use in sites requiring fast test results and limited sample preparation.

A Novel Electrochemical Assay System

This chapter presents an overview of a novel electrochemical assay system. Electrochemical Cloned Enzyme Donor ImmunoAssay (ECEDIA) integrates the CEDIA technology developed by Microgenics with the electrochemical sensor platform developed at Boehringer Mannheim. Theophylline is the analyte. Recombinant DNA technology is utilized to produce a unique homogeneous enzyme immunoassay system. The B-galactosidase enzyme has been split into two inactive fragments. Enzyme Acceptor (EA) is a large fragment containing approximately 95% of the native enzyme, the other fragment Enzyme Donor (ED). EA can spontaneously recombine with ED to form a catalytically active B-galactosidase enzyme. Theophylline has been covalently attached to the ED in a way that does not interfere with the reassociation of the enzyme fragments to form an active enzyme. Theophylline specific antibody, when bound to the Ed-theophylline conjugate, regulates the amount of enzyme formed by inhibiting the reassociation of the ED and EA fragments. Theophylline in the test sample competes for the specific anti-theophylline binding sites. The amount of enzyme which re-associates is directly proportional to the concentration of theophylline in the sample.