Dataset Information

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10.

ABSTRACT:

Objective

M10 is a ten amino acid peptide generated from the intracellular cytoplasmic tail of the hepatocyte growth factor (HGF) receptor c-Met following cleavage by caspase-3. Recently we reported that M10 interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo and can be advanced into a novel antifibrotic remedy. The current study was undertaken to develop an immunoassay to measure M10 concentration in biological specimens.

Experimental design

An Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for detection of M10 in biological fluids was developed using pharmaceutical grade synthetic M10 as a calibrator and commercially available anti-c-Met C12 antibody.

Results

M10 ELISA specifically detected in plasma M10, but not a scrambled peptide, following a single intraperitoneal administration of M10 (1mg/kg) to mice. The detection limit was 9.6 ng/ml, and the measuring limit was between 15 ng/ml and 200 ng/ml. The recovery limits of M10 were between 80% and 120%; intra-assay coefficient of variation was between 5.3% and 6.3%; inter-assay coefficient of variation was between 5.0% and 8.0% over the buffer concentration tested in the range from 15 ng /ml to 250 ng /ml. The peak of M10 concentration following a single intraperitoneal injection (1mg/kg) was achieved within 6 hours and declined to minimal levels by 48 hours. The experimentally obtained half-life for M10 was comparable to the theoretically predicted half-life for M10.

Conclusions

We have established a highly sensitive ELISA to detect the antifibrotic peptide M10 in plasma samples, which should prove to be a novel tool to study the pharmacokinetics and efficacy of M10 in the treatment of fibroproliferative disorders.

SUBMITTER: Akter T

PROVIDER: S-EPMC5703532 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10.

Akter Tanjina T Atanelishvili Ilia I Noguchi Atsushi A Silver Richard M RM Bogatkevich Galina S GS

PloS one 20171127 11

<h4>Objective</h4>M10 is a ten amino acid peptide generated from the intracellular cytoplasmic tail of the hepatocyte growth factor (HGF) receptor c-Met following cleavage by caspase-3. Recently we reported that M10 interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo and can be advanced into a novel antifibrotic remedy. The current study was undertaken to develop an immunoassay to measure M10 concentration in biological specimens.<h4>Experimental design</h4>An Indi ...[more]

PMID: 29176766

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Project description:S. suis is an important zoonotic pathogen from sick and recessive carrier pigs that poses a serious threat to animal husbandry production and public health. It usually causes horizontal transmission among pigs. The morbidity and mortality of this disease are very high. Human infection is caused through direct or indirect contact with sick pigs. The two large-scale outbreaks in China were due to the outbreak of S. suis on pig farms, which spread to human infection; thus, detecting S. suis in pig herds is crucial. At present, the commercial S. suis ELISA type 2 kits on the market can only detect single serotypes, high probabilities of interaction reactions, and biosafety risks when using inactivated S. suis as an antigen. Phosphate-3-glyceraldehyde dehydrogenase (GAPDH), muramidase-released protein (MRP), and dihydrolipoamide dehydrogenase (DLDH) are important S. suis type 2, S. suis type 7, and S. suis type 9 protective antigens. This study purified the GMD protein (B-cell-dominant epitopes of GAPDH, MRP, and DLDH antigens) and used a diverse combination of dominant epitopes of the multiple different antigens as coated antigens, improving the sensitivity and safety of the indirect ELISA experiments. An indirect ELISA method (GMD-ELISA) was developed for detecting S. suis antibodies. The antigen-antibody response was optimized using checkerboard titration. The results of testing using ELISA for Salmonella enterica (S. enterica), Escherichia coli (E. coli), Staphylococcus aureus (SA), and Streptococcus pyogenes (S. pyogenes) were all negative, indicating that this method had strong specificity. The results were still positive when the dilution ratio of S. suis-positive serum reached 1:6, 400, thus indicating that the method had high sensitivity. The results of the reproducibility assay for indirect ELISA showed that the intra-assay coefficient of variation and the inter-assay coefficient of variation were less than 10%, indicating that the method had good repeatability. We investigated the seroprevalence of S. suis in 167 serum samples collected in East China, and 33.5% of the samples were positive for antibodies against S. suis, indicating that the prevalence of S. suis is high in pig farms in Eastern China. The novel GMD-ELISA is a convenient, sensitive, and specific diagnostic method that provides technical support for rapid diagnosis and epidemiological investigation.

Dataset Information

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10.

Objective

Experimental design

Results

Conclusions

Publications

Establishment of an indirect ELISA for detection of the novel antifibrotic peptide M10.

Similar Datasets

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