Hiroko Tohge

An enzyme- linked immunosorbent assay for heparan sulfate proteoglycans

An enzyme- linked immunosorbent assay (ELISA) for heparan sulfate proteoglycan (HSPG) was developed based on the high affinity binding profile of HSPG to lipoprotein lipase (LPL). LPL was shown to bind to precoated HSPG in dose dependent manner and was determined spectrophotometrically using specific anti- LPL antibody. This ELISA allowed to evaluate HSPG produced by PC12 cell with clear linearity at range of 10 - 500 ng/ml. Soluble chondroitin sulfate proteoglycan (CSPG) from rat brain, which was not detectable by this method, did not exhibit any inhibitory effects on affinity binding of HSPG to LPL, even if 8 times higher concentrations of CSPG to HSPG was added. The sensitivity of this ELISA was about 100 times higher than that of conventional carbazole reaction method. These findings indicated its potential usefulness of this method for measuring small amounts of HSPG capable of binding to LPL and for studying biological implications of HSPG - LPL interaction.

Immunofixation electrophoretogram of the third component of complement in human serum by the use of new barbital·lithium·HCl buffer on agarose gel

Separation and measurement of the third component of complement (C3) and its conversion products in normal human serum can be achieved using the immunofixation electrophoretic technique on agarose gel with the barbltal·lithium·HCl buffer (pH 8.6, μ0.1) firstly proposed by M. Ichimura and T. Baba.The C3 immunofixation electrophoresis worked well, because utilization of this new buffer was effective on highly clear separation of the β-positioned serum proteins. Consequently, we have resolved native C3 and six C3 conversion products and simultaneously obtained the C3 conversion sequence in serum.In addition, we are able to measure the concentrations of serum C3 on its conversion products and the C3 biological activities by means of the immunofixation electrophoresis.