Drago, Guido Andre; (1992) The development of ultrasensitive immunological methods for the detection of protein polymorphisms. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the development of ultrasensitive methods for the immunological detection of two highly polymorphic human proteins, group-specific component (GC) and phosphoglucomutase-1 (PGM) following separation by high resolution isoelectric focusing (IEF) and transfer to immobilising membranes. All the steps involved in the immunoblotting procedure were investigated and the procedure optimised with a view to its application in the field of Forensic Science. In the case of the GC polymorphism, a 3000 fold increase in the sensitivity of detection was achieved compared with immunofixation methods. The improved immunoblotting procedure allowed the phenotyping of GC from forensic bloodstains, semen stains and post-coital vaginal swabs. A high titre sheep anti-rabbit muscle PGM antiserum was produced that was capable of detecting both rabbit and human PGMl isozymes on immunoblots, following sodium-dodecyl-sulphate gel electrophoresis or IEF, of tissues expressing high levels of the enzyme. This antiserum was used successfully for the detection of the human PGMl polymorphism in fetal tissue from forensic casework samples, and the detection of low levels of PGM activity in rabbit haemolysates but was not suitable for screening human haemolysate PGM. The antiserum did not bind either PGM2 or PGM3 isozymes, but recognised the milk (PGM4) isozymes. This led to detailed studies of the biochemical and genetical features of the milk PGM isozymes and a novel method for screening the PGM2 isozyme polymorphism. The sheep anti-rabbit PGM was used to purify small amounts of human PGMl from skeletal muscle for use as an immunogen. An alternative method for producing the PGM protein, using cloned protein DNA sequence and a lambda gtll expression vector was also investigated.

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