The discrepancy between the cross-reactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity.

The rat monoclonal antibody YC5/45 has been shown to react specifically with serotonin containing neurones, and does not detect dopamine containing sites. On the other hand the monoclonal antibody recognizes dopamine, since it is capable of inhibiting the binding of 3H-YC5/45 to sheep red blood cells coated with serotonin albumin conjugate, or the direct agglutination of the same cells by the monoclonal antibody. Tryptamine and 5-methoxytryptamine are even better inhibitors, while derivatives with a modified-CH2-CH2-NH2 group are inactive. Treatment of the active compounds with paraformaldehyde increases their efficiency dramatically. The binding of antibody was also tested by cross linking with paraformaldehyde: dopamine, serotonin, tryptamine, 5-methoxytryptamine etc., to serum albumin. Only the serotonin derivative was active. The result shows that the discrimination specificity observed in immunohistochemistry is due to the paraformaldehyde fixation. It is concluded that for histochemical detection of serotonin by YC5/45, two reactive sites are necessary. The -CH2-CH2-NH2 group is probably modified by formaldehyde to produce a cyclic derivative to form the antibody recognition site. A different formaldehyde reactive site is required for cross linking to the tissue and this is provided by the presence of the 5-OH of serotonin which is absent in the others.