A new ELISA method to identify the D327N mutation of human sex hormone binding globulin (SHBG)

Abstract

The D327N mutation of human SHBG is associated with a number of good prognostic factors in breast cancer like estrogen receptor positivity and erb2 negativity. The identification of this mutation, that requires only a small sample of circulating blood, could be helpful whenever tissue samples are too scanty for the determination of prognostic factors, e.g. at fine needle aspiration for cytology. The search for this mutation is routinely performed in our laboratory with the HinfI restriction fragment length polymorphism (RFLP) technique on polymerase chain reaction (PCR)-amplified DNA. The present report describes a new and simple enzyme-linked immunosorbent assay (ELISA) method to detect the SHBG mutation. The DNA enzyme immuno assay (DEIA) method, that is widely used in virology and has already been used to identify single nucleotide mutations, allows the identification of hybrids between specific DNA sequences and biotynilated probes with a monoclonal antibody against double stranded-DNA. We here report that by using two specific probes, specifically built for this kind of test, one complementary to the wild type SHBG sequence and the other to the D327N mutated DNA, the DEIA technique can be used to identify on PCR-amplified DNA the D327N mutation of human SHBG exactly as the HinfI RFLP technique. The DEIA technique could, thus, be especially helpful when a large number of samples has to be processed, the technique being easier than HinfI RFLP, specific, reproducible and allowing substantial time saving.