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Characterization of degradation process of sucrase-isomaltase in rat jejunum with monoclonal-antibody-based enzyme-linked immunosorbent assay.

ABSTRACT: As shown previously, during degradation of sucrase-isomaltase in rat jejunum, degradation of the sucrase active site occurs before that of isomaltase active site [Goda & Koldovský (1985) Biochem. J. 229, 751-758]. To characterize further the process of sucrase-isomaltase degradation in jejunum, we determined the amounts of immunoreactive sucrase-isomaltase in rat jejunum by using a monoclonal-antibody-based enzyme-linked immunosorbent assay. By employing two alternative monoclonal antibodies (one reacting with the sucrase subunit and the other reacting with the isomaltase subunit), the amount of antigen-containing sucrase subunit and the amount of antigen-containing isomaltase subunit were separately quantified. In both upper and lower jejunum of rats, the amount of antigen-containing isomaltase subunit was always higher than the amount of antigen-containing sucrase subunit. This difference was attributable mainly to a degradation product of sucrase-isomaltase, which was identified as isomaltase monomer. Occlusion of pancreatic ducts for 18 h eliminated the difference between the amount of antigen-containing sucrase subunit and the amount of antigen-containing isomaltase subunit in both upper and lower jejunum. In jejunum of control animals, the molar ratio of sucrase subunit to isomaltase subunit was estimated to be 0.32-0.52, indicating that quite a large proportion of sucrase-isomaltase (48-68%) is present as degradation products (e.g. isomaltase monomer). These results support the model of degradation process of sucrase-isomaltase in brush-border membranes of rat jejunum, whereby degradation of sucrase subunit by the action of pancreatic proteinase(s) precedes degradation of isomaltase subunit.

SUBMITTER: Goda T

PROVIDER: S-EPMC1148811 | biostudies-other | 1988 Feb

REPOSITORIES: biostudies-other

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Project description:BackgroundCassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA).MethodsMonoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard.ResultsA TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA.ConclusionsOur findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

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Characterization of degradation process of sucrase-isomaltase in rat jejunum with monoclonal-antibody-based enzyme-linked immunosorbent assay.

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