Elisa of an inhibitor of tissue-type plasminogen activator from Erythrina caffra Thunb.

Abstract

An Elisa for an inhibitor of tissue-type plasminogen activator (t-PA) from Erythrina caffra Thunb. was developed using polyclonal antibodies. The optimal antibody concentration used to coat the wells was 6 mg l− 1. Tween 20 in the incubation and washing buffers reduced the non-specific binding to the wells notably and was more effective than blocking with bovine serum albumin. Rinsing the plates three to six times had no significant effect on the efficiency of the assay. The optimum incubation period for the assay was 60 min. Large differences were found in the sensitivity of the assay with the use of different makes of microtiter plates. The t-PA inhibitor was detected in the leaves of 24 species belonging to the genus Erythrina. Large differences in the t-PA inhibitor levels were found between species but no correlation was found between the t-PA inhibitor content of the species and their taxonomical sections or their geographical distribution. The t-PA inhibitor was not found in members of other genera of the Leguminosae such as Glycine max (L.) Merrill, Pisum sativum L. and Phaseolus vulgaris L. ’n Ensiemgekoppelde immunosorbenttoets (Elisa) is met poliklonale teenliggame ontwikkel vir ’n inhibeerder van weefselplasminogeenaktiveerder (t-PA) wat in Erythrina caffra Thunb. voorkom. Die optimale teenliggaamkonsentrasie vir die bedekking van die mikrotiterplaat was 6 mg l− 1. Tween 20 in die inkuberings- en wasbuffers het die nie-spesifieke binding aansienlik verlaag en was meer doeltreffend as ’n blokkeringstap met beesserumalbumien. Die doeltreffendheid van die immunotoets is nie beïnvloed deur die mikrotiterplate drie tot ses keer tussen die inkuberingstappete was nie. Die optimale inkuberingsperiode vir die toets was 60 min. Mikrotiterplate van verskillende vervaardigers het ’n aansienlike invloed op die sensitiwiteit van die toets gehad. Die t-PA-inhibeerder is in die blare van 24 Erythrina spesies wat getoets is, gevind. Groot verskille in die t-PA-inhibeerdervlakke is tussen die spesies gevind, maar geen korrelasie is gevind tussen die t-PA-inhibeerdervlakke van die spesies en hul taksonomiese seksies of hul geografiese verspreiding nie. Die t-PA-inhibeerder is nie in verteenwoordigers van ander genera van die Leguminosae, soos Glycine max (L.) Merrill, Pisum sativum L. en Phaseolus vulgaris L., gevind nie.