Molecular detection of methionine in rat brain using specific antibodies

In order to study the localization of methionine in rat brain, an immunological approach was developed by raising antibodies directed against this amino acid. Methionine was conjugated to bovine serum albumin (BSA) or human serum albumin (HSA) via glutaraldehyde. The conjugates were then reduced by sodium borohydride and injected alternately into rabbits. Antibody affinity and specificity were evaluated using an adapted ELISA method, by competition experiments between conjugated methionine and related conjugated compounds, pre-incubated with anti-methionine antibodies diluted at 1/20,000. The resulting cross-reactivity ratios, calculated at half-displacement, showed that glutaraldehyde-methionine conjugate (methionine-G-BSA) was the best recognized compound. Non-reduced methionine conjugate (methionine=G=BSA) and the related-conjugated molecules such as homocysteine, homocysteic acid, cysteine, cystathionine and glutamate were not recognized at all. Antibodies to methionine were directed against a glutaraldehyde-methionine epitope and their very high affinity and specificity made them reliable tools for molecular detection of methionine in rat brain. Using purified antibodies diluted at 1/20,000, motoneurons were found to be the most methionine-immunoreactive cell bodies in glutaraldehyde-fixed rat brain sections.