Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: evidence that transporter undergoes endoproteolytic cleavage.

Background: Serotonin transporter (SERT) is important target molecule for many antidepressive drugs and substances of abuse and is implicated in psychiatric disorders. We performed immunoblotting analysis of human and rat SERT in platelets and brain using the panel of eight site-specific SERT monoclonal and polyclonal antibodies (mAbs and pAbs).

Methods: SDS-PAGE/Western blotting was conducted using peroxidase-labeled DEAE and affinity purified SERT antibodies under conditions preventing SERT post-extraction degradation.

Synergistic effects in antigen capture ELISA using three monoclonal antibodies directed at different epitopes of the same antigen.

Using a panel of monoclonal antibodies (mAb) against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture enzyme-linked immunosorbent assay (ELISA) may be significantly increased by the simultaneous immobilization on a solid phase of two co-operating capture mAbs. This method ("a three-site ELISA") uses three mAbs at different epitopes of the same antigen (two capture/one tracer), unlike the traditional two-site assay, using one capture and one tracer mAbs. We established two-site and three-site ELISA assays for Mb, by varying capture and tracer mAbs.

Triple-site antigen capture ELISA for human myoglobin can be more effective than double-site assay.

Using a panel of monoclonal antibodies against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture ELISA can be significantly increased by simultaneous immobilization of two cooperating capture monoclonal antibodies on a solid phase. This method ("triple-site ELISA") uses three monoclonal antibodies to different epitopes of the same antigen (two capture/one tracer) unlike the traditional double-site assay using one capture and one tracer monoclonal antibody. We developed double- and triple-site ELISA for Mb by varying the capture and tracer monoclonal antibodies.

Single-step sandwich immunoassay of myoglobin with bifunctional monoclonal antibodies.

Two protocols for sandwich antigen-capture ELISA of human myoglobin were compared. In the first (routine) variant, 14D6 monoclonal antibodies conjugated to horseradish peroxidase were used as the secondary antibodies. Bifunctional antibodies specific for myoglobin/peroxidase were used as the secondary antibodies in the second variant.