Measurement of an analog of insulin-like growth factor-I in blood plasma using a novel enzyme-linked immunosorbent assay

Long-Arg³-IGF-I (LR³IGF-I) is a synthetic analog of IGF-I that has much lower affinity for IGF-binding proteins than do native IGFs-I and -II. Comparisons of the effects of LR³IGF-I with those of IGFs-I and -II in in vitro and in vivo studies have proved useful in defining the functions of IGF-binding proteins. We have developed a sensitive noncompetitive nonisotopic assay of LR³IGF-I. Mouse IgG 1A7–F5–E5 binds an epitope that contains the substituted arginine³ in LR³IGF-I and was used as the solid phase antibody. The solution phase antibody was a rabbit immunoglobulin Nelson which binds to an epitope that is common to IGF-I and LR³IGF-I. The ELISA system was able to detect as little as 50 pg LR³IGF-I in 100 μl and the native peptides IGFs-I and -II have less than 0•01% activity. Blood plasma from animals treated with pharmacologically active doses of this growth factor analog could be diluted 33•3-fold before assay, at which concentrations plasma had no significant effect on the assay. The ELISA response to LR³IGF-I was unaffected by the presence of IGF-binding proteins. The intra-assay and interassay coefficients of variation are 2•8 and 7•3% respectively. Recovery of LR³IGF-I added to blood plasma was 90%. The ELISA was used to measure LR³IGF-I concentrations in plasma of cows treated with a pharmacologically active dose of this peptide and the results were compared with those obtained by a previously established LR3IGF-I RIA that requires size exclusion chromatography of plasma under acidic conditions to eliminate IGF-binding protein artefacts from the RIA. There was a positive correlation between results obtained by the two assays. The LR³IGF-I ELISA permits discrimination between the exogenous synthetic IGF-I analog and the endogenous native IGFs-I and -II in animals treated with this growth factor without the need for radioiodination of LR³IGF-I and elimination of the requirement for extraction of plasma before assay.O Gajanandana, K Irvine, PA Grant, GL Francis, SE Knowles, J Wrin, JC Wallace, and PC Owen

Epidemiology of Tospoviruses in South and Southeast Asia: Current status and future prospects

Tospoviruses are emerging as a major constraint to the production of a broad range of economically important crops in South and Southeast Asia (S & SEA). Available data suggest that the majority of these viruses belong to serogroup IV (Watermelon silver mottle virus serogroup). Many of these viruses show geographical structuring in that they are restricted to the Asian continent. In recent years, tospovirus species like Capsicum chlorosis virus and Iris yellow spot virus, present in other continents, have been reported in the S & SEA region. Among different species of thrips (Thysanoptera: Thripidae) that have been confirmed as vectors of one or more tospoviruses worldwide, only a few have been authenticated to be present in the S & SEA region. The current knowledge on the distribution of thrips vectors and tospoviruses in the region will be reviewed and the research needs for a better understanding of the growing impact of tospoviruses and thrips vectors will be discussed

Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection

Strategies to improve the surface plasmon resonance-based immmunodetection of bacterial cells

We have made a comparison of (a) different surface chemistries of surface plasmon resonance (SPR) sensor chips (such as carboxymethylated dextran and carboxymethylated C1) and (b) of different assay formats (direct, sandwich and subtractive immunoassay) in order to improve the sensitivity of the determination of the model bacteria Acidovorax avenae subsp. citrulli (Aac). The use of the carboxymethylated sensor chip C1 resulted in a better sensitivity than that of carboxymethylated dextran CM5 in all the assay formats. The direct assay format, in turn, exhibits the best sensitivity. Thus, the combination of a carboxymethylated sensor chip C1 with the direct assay format resulted in the highest sensitivity for Aac, with a limit of detection of 1.6x106 CFU mL-1. This SPR immunosensor was applied to the detection of Aac in watermelon leaf extracts spiked with the bacteria, and the lower LOD is 2.2x107 CFU mL-1