Selective recognition of M235T angiotensinogen variants and their determination in human plasma by monoclonal antibody-based immunoanalysis

The common M235T mutation of human angiotensinogen has been shown to be associated with a 10-20% increase in plasma angiotensinogen level and increased frequency of essential and pregnancy-induced hypertension. The detection of such a common factor in the plasma of individuals at risk could be a useful tool for modern molecular-based medicine. The recognition of M235T variants was investigated using four monoclonal antibodies (mAbs) directed against human angiotensinogen; two immunometric assays were developed. The first assay (using mAbs 7B2 and 4G3) allowed the direct determination of angiotensinogen concentrations and did not show a significant difference with the enzymatic measurement of angiotensinogen. The second assay (using mAbs 1H8 and 1C11) showed a fine distinction between the T235 mutant and M235 wild-type forms of angiotensinogen, with a greater affinity for the latter, as confirmed by biosensor BIAcore experiments. This assay was extremely sensitive in measuring the proportions of the M235 and T235 forms present in the test samples, the first time such a distinction has been achieved in the serpin family. The simple immunoanalysis of the plasma allowed the direct determination of the M235T genotype of the individual tested. Furthermore, it was shown that the T174M mutation, described as being in complete linkage disequilibrium with the M235T mutation, had no influence on these results. Moreover, this assay suggested the presence of the M235 and T235 angiotensinogens in approximately equal amounts in heterozygous plasmas. In conclusion, the immunometric assays described in this study should provide original tools for investigating the relationship between M235T genotype, plasma angiotensinogen levels, and regulation of blood pressure.