2 research outputs found
An overall evaluation of the resistance (R) and pathogenesis-related (PR) superfamilies in soybean, as compared with Medicago and Arabidopsis
Detection of proteases using an immunochemical method with haptenylatedâgelatin as a solid-phase substrate.
A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatinâhapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase proteinâhapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatinâcholic acid conjugates, affinity-purified sheep anti-cholic acid antibodyâHRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Doseâresponse curves for enzyme activities were measured within ranges of 0â550 ”units mLâ1 for chymotrypsin, 0â12 ”units mLâ1 for type IX, 0â35 ”units mLâ1 for type XIV and 0â100 ”units mLâ1 for type XXIV. The detection limits of the proteases studied were 89 ”units mLâ1 for chymotrypsin, 0.26 ”units mLâ1 for type IX, 5.8 ”units mLâ1 for type XIV and 6.5 ”units mLâ1 for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format