The use of the RACE method to clone hybridoma cDNA when V region primers fail

Abstract

The technique of V region PCR to clone antibody V regions from hybridomas has been extensively used. However, in addition to, or even instead of, cloning the V regions with the desired specificity, myeloma cell derived V regions, V regions which are the result of non-productive rearrangements, and also V regions which are productive but which do not recognise the antigen of interest, may be isolated. In this paper we describe a comparison of the use of V region PCR and a modification of the RACE technique to clone the V region of the anti-NGF hybridoma, alpha D11. This hybridoma has heavy and light chain V regions which are refractory to amplification with V region primers, but which are easily amplified using RACE, a PCR based procedure which is independent of the variability within the V regions.