Characterization of a monoclonal antibody specific to a flavonol 2′-O-glucosyltransferase

Abstract

A monoclonal antibody to a partially purified preparation of 2'-O-glucosyltransferase was produced by in vitro immunization of spleen cells from BALB/c mice, followed by fusion with mouse myeloma cells. Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay for (i) their ability to produce immunoglobulins and (ii) their immunoreactivity with a partially purified enzyme preparation. The majority of the immunoglobulin-producing hybridomas were IgM secretors. Two highly immunoreactive IgM-secreting clones were chosen for further characterization. The supernatant fraction from a culture of one of these clones displayed 50% inhibition of the 2'-O-glucosyltransferase activity. The native form of the 2'-O-glucosyltransferase was essential for recognition, suggesting that the epitope recognized by the antibody is a conformational discontiguous one.

A forty-year journey in plant research: original contributions to flavonoid biochemistry

“…A highly immunoreactive IgM-secreting clone displays >50% inhibition of the 2′-GT, but not the 5′-GT, activity. Moreover, the native form of the enzyme is essential for antibody recognition on Western blot analysis (Latchinian and Ibrahim 1989). The above account indicates that the enzymatic synthesis of polymethylated flavonol glucosides in C. americanum is regulated by strict substrate and position specificities of the enzymes involved, their ordained sequence in the pathway, and some kinetic considerations.…”

Immunocytochemical localization of plant secondary metabolites and the enzymes involved in their biosynthesis

“…Very recently, however, a monoclonal antibody to a partially purified flavonol 2'/S'-O-glucosyltransferase was produced in our laboratory by an in oitro immunization technique (Boss, 1986). This antibody displayed significant inhibition of activity of the 2'-, but not of the 5'-0-glucosyltransferase activity, thus indicating the involvement of two distinct enzymes in the glucosylation of the 2'and 5'-positions of partially methylated flavonols (Latchinian el ul., 1987;Latchinian and Ibrahim, 1989). Furthermore, the fact that this antibody could not recognize its respective antigen in immunolocalization studies (Latchinian-Sadek and Ibrahim, 1990) implied recognition o f an asscmbled topographic epitope, rather than a sequential contiguous onc (Berkofsky, 1981).…”