Preparation of monoclonal antibody against d-3-methoxy-4-hydroxyphenylglycol

Abstract

We have been trying to prepare a specific monoclonal antibody against an endogenous 3-methoxy-4-hydroxyphenylglycol (MHPG) which will be a diagnostic marker for mental diseases such as depression. In our previous paper, we discriminated d- and l-MHPG separated from its racemate by chiral chromatography judging from their optical rotations and retention times in comparison with those of purified d-MHPG from human urine. In this paper, a d-MHPG-specific monoclonal antibody was prepared to develop an enzyme immunoassay (EIA). An adequate amount of d-MHPG as hapten was purified by chiral chromatography using a Ceramospher Chiral RU-1 column. The hapten was conjugated to human serum albumin as a carrier protein by a Mannich reaction with formaldehyde. This antigen solution was mixed with an equal volume of Freund's complete adjuvant and injected into 3 WKY/NCrj rats. After the titer of antisera increased, medial iliac lymph node cells from a rat in the highest titer were fused with SP2/0 myeloma cells. Hybridomas were screened by competitive EIA remarking recognition of dl-MHPG and cloned by a limited dilution method. After the fourth cloning, a monoclonal cell line was established. The monoclonal antibody in a culture medium was examined on EIA with 25 competitors including d-MHPG. Nineteen analogs showed little cross reactivity. Only 5 compounds showed weak cross reactivity. This concluded that the monoclonal antibody was reasonably specific for d-MHPG and should be useful for practical EIA.