Efficient generation of monoclonal antibodies for specific protein domains using recombinant immunoglobulin fusion proteins: pitfalls and solutions.

摘要

Monoclonal antibody production (mAb) first requires the availability of large amounts of pure immunogen for animal immunisation and fusion screening procedures. To overcome this obstacle, we have developed a simple method for rapid generation of pure antigen by generation of recombinant protein containing the antigen of interest fused to the hinge and Fc domains of human immunoglobulin (Ig). The Fc domain forms a convenient 'tag' to enable detection of the protein in supernatant of transfected cells and for purification of immunogen by protein A affinity chromatography. The only requirement for immunogen preparation using this methodology is that a DNA sequence encoding a portion of the molecule of interest is known and that a suitable PCR template is available. Antibody production can be tailored to specific protein domains, for example functional domains, by expressing solely those domains in the fusion protein. We illustrate the technique with two different fusions used to raise antibodies against the porcine and human analogues of a complement (C) regulatory protein, decay accelerating factor (DAF) (CD55). Use of the specific Ig-fusion protein and a control protein facilitated screening of fusions by ELISA. We demonstrate two expression systems used to generate Ig fusion proteins, the first utilised a commercial vector to incorporate an amino terminal leader sequence and carboxy terminal Ig domains. Low levels of expression required subcloning into a high expression vector and resulted in yields of fusion protein at between 2 and 10 mg per litre of supernatant. The second expression system utilised the high expression vector directly, Ig domains of the chosen immunoglobulin isotype were amplified from peripheral blood mononuclear cell (PBMC) RNA and ligated into the vector in frame with DNA encoding the antigen. We describe potential pitfalls that may be encountered while using Ig fusion proteins as immunogen and demonstrate ways in which to tailor their design for optimal mAb production.