scholarly journals Rapid Differential Detection of Abrin Isoforms by an Acetonitrile- and Ultrasound-Assisted On-Bead Trypsin Digestion Coupled with LC-MS/MS Analysis

Toxins ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 358
Author(s):  
Long-Hui Liang ◽  
Yang Yang ◽  
Shu Geng ◽  
Xi Cheng ◽  
Hui-Lan Yu ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Polyclonal Antibodies ◽  
Lethal Dose ◽  
Gel Filtration ◽  
Trypsin Digestion ◽  
Differential Detection ◽  
Digestion Method ◽  
Complex Matrices ◽  
Protein Toxin ◽  
Ultrasound Assisted

The high toxic abrin from the plant Abrus precatorius is a type II ribosome-inactivating protein toxin with a human lethal dose of 0.1–1.0 µg/kg body weight. Due to its high toxicity and the potential misuse as a biothreat agent, it is of great importance to developing fast and reliable methods for the identification and quantification of abrin in complex matrices. Here, we report rapid and efficient acetonitrile (ACN)- and ultrasound-assisted on-bead trypsin digestion method combined with HPLC-MS/MS for the quantification of abrin isoforms in complex matrices. Specific peptides of abrin isoforms were generated by direct ACN-assisted trypsin digestion and analyzed by HPLC-HRMS. Combined with in silico digestion and BLASTp database search, fifteen marker peptides were selected for differential detection of abrin isoforms. The abrin in milk and plasma was enriched by immunomagnetic beads prepared by biotinylated anti-abrin polyclonal antibodies conjugated to streptavidin magnetic beads. The ultrasound-assisted on-bead trypsin digestion method was carried out under the condition of 10% ACN as denaturant solvent, the entire digestion time was further shortened from 90 min to 30 min. The four peptides of T3Aa,b,c,d, T12Aa, T15Ab, and T9Ac,d were chosen as quantification for total abrin, abrin-a, abrin-b, and abrin-c/d, respectively. The absolute quantification of abrin and its isoforms was accomplished by isotope dilution with labeled AQUA peptides and analyzed by HPLC-MS/MS (MRM). The developed method was fully validated in milk and plasma matrices with quantification limits in the range of 1.0-9.4 ng/mL for the isoforms of abrin. Furthermore, the developed approach was applied for the characterization of abrin isoforms from various fractions from gel filtration separation of the seeds, and measurement of abrin in the samples of biotoxin exercises organized by the Organization for the Prohibition of Chemical Weapons (OPCW). This study provided a recommended method for the differential identification of abrin isoforms, which are easily applied in international laboratories to improve the capabilities for the analysis of biotoxin samples.

scholarly journals Streptococcus salivarius Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure

2001 ◽  
Vol 183 (9) ◽  
pp. 2724-2732 ◽  
Author(s):  
Céline Lévesque ◽  
Christian Vadeboncoeur ◽  
Fatiha Chandad ◽  
Michel Frenette
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Surface ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Spontaneous Mutant ◽  
Streptococcus Salivarius ◽  
Streptococcal Species ◽  
Repeated Motif

ABSTRACT Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivariusfimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 106 to 40 × 106Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis andStreptococcus constellatus.

Can Serum Thymidine Kinase 1 Detect Small Invisible Malignant Tumours?

2021 ◽  
Author(s):  
Ji Zhou ◽  
Huijun Li ◽  
Cong Fang ◽  
Junye Tan ◽  
Peng Gao ◽  
...  
Keyword(s):  
Thymidine Kinase ◽  
Total Protein ◽  
Magnetic Beads ◽  
Polyclonal Antibodies ◽  
Malignant Tumour ◽  
The Body ◽  
Cell Diameter ◽  
Malignant Tumours ◽  
Thymidine Kinase 1 ◽  
Protein Cell

Abstract Objectives. Early detection of malignant tumour is a prerequisite for a successful treatment. Here we investigate if thymidine kinase 1 is more sensitive than imaging technology to discover small invisible malignant tumours.Material and Methods. The cellular concentration of TK1 was determined by a novel automatic chemiluminescence analyzer of magnetic particle immune sandwich minimum. The primary and secondary antibodies linked to the magnetic beads were chicken anti-human thymidine kinase 1 IgY-polyclonal antibodies (IgY pAb). The minimum number of cells able to be detected by the novel detection technology using an automatic chemiluminescence analyzer were determined based on the cellar TK1 concentration of low and high TK1 cell lines of known cell count.Results. The TK1 concentration of malignant cell was found to be 0.021 pg/cell. Assuming 200 pg of total protein/cell, TK1 corresponds to 0.01 % of the total protein/cell. The concentration of TK1 in human blood serum of malignant patients is in the range of 2-10 pmol/l (pM), corresponding to about 50 x106 growing cells in the body that release TK1 into 5 litre blood. The limit visibility by imaging of a tumour is about 1 mm in diameter, corresponding to about 109cells of a cell diameter of 1µm. Conclusion. TK1 is more sensitive than imaging.

scholarly journals Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes

1992 ◽  
Vol 287 (2) ◽  
pp. 443-446 ◽  
Author(s):  
O A Coso ◽  
A Díaz Añel ◽  
H Martinetto ◽  
J P Muschietti ◽  
M Kazanietz ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Pertussis Toxin ◽  
Polyclonal Antibodies ◽  
Gel Filtration ◽  
Binding Activity ◽  
Gi Protein ◽  
Liver Membranes ◽  
Blocking Capacity ◽  
Polypeptide Band

A guanosine 5′-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.

Direct Acetonitrile-Assisted Trypsin Digestion Method Combined with LC–MS/MS-Targeted Peptide Analysis for Unambiguous Identification of Intact Ricin

2020 ◽  
Vol 20 (1) ◽  
pp. 369-380 ◽  
Author(s):  
Chang-Cai Liu ◽  
Long-Hui Liang ◽  
Yang Yang ◽  
Hui-Lan Yu ◽  
Long Yan ◽  
...  
Keyword(s):  
Trypsin Digestion ◽  
Peptide Analysis ◽  
Digestion Method

Myxosoma cerebralis: Isolation and Concentration from Fish Skeletal Elements — Trypsin Digestion Method

1973 ◽  
Vol 30 (11) ◽  
pp. 1713-1716 ◽  
Author(s):  
Marsha L. Landolt
Keyword(s):  
Spore Suspension ◽  
Trypsin Digestion ◽  
Improved Method ◽  
Mechanical Grinding ◽  
Digestion Method ◽  
Skeletal Tissue

An improved method for detection of Myxosoma cerebralis spores is described. Spores were freed from skeletal tissue by trypsin digestion and ether was used for purification of spore suspension. As compared to mechanical grinding, this procedure resulted in a sixfold increase in observable spores.

scholarly journals Gr1+ Cells Control Growth of YopM-Negative Yersinia pestis during Systemic Plague

2009 ◽  
Vol 77 (9) ◽  
pp. 3791-3806 ◽  
Author(s):  
Zhan Ye ◽  
Edward J. Kerschen ◽  
Donald A. Cohen ◽  
Alan M. Kaplan ◽  
Nico van Rooijen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Yersinia Pestis ◽  
Nk Cell ◽  
Early Stage ◽  
Lethal Dose ◽  
Cell Types ◽  
Growth Defect ◽  
Protein Toxin ◽  
Inflammatory Monocytes ◽  
Control Growth

ABSTRACT YopM, a protein toxin of Yersinia pestis, is necessary for virulence in a mouse model of systemic plague. We previously reported YopM-dependent natural killer (NK) cell depletion from blood and spleen samples of infected mice. However, in this study we found that infection with Y. pestis KIM5 (YopM+) caused depletion of NK cells in the spleen, but not in the liver, and antibody-mediated ablation of NK cells had no effect on bacterial growth. There was no YopM-associated effect on the percentage of dendritic cells (DCs) or polymorphonuclear leukocytes (PMNs) in the early stage of infection; however, there was a YopM-associated effect on PMN integrity and on the influx of monocytes into the spleen. Ablation of Gr1+ cells caused loss of the growth defect of YopM− Y. pestis in both the liver and spleen. In contrast, ablation of macrophages/DCs inhibited growth of both parent and mutant bacteria, accompanied by significantly fewer lesion sites in the liver. These results point toward PMNs and inflammatory monocytes as major cell types that control growth of YopM− Y. pestis. Infection with fully virulent Y. pestis CO92 and a YopM− derivative by intradermal and intranasal routes showed that the absence of YopM significantly increased the 50% lethal dose only in the intradermal model, suggesting a role for YopM in bubonic plague, in which acute inflammation occurs soon after infection.

scholarly journals Detection of Botulinum Neurotoxin Serotype A, B, and F Proteolytic Activity in Complex Matrices with Picomolar to Femtomolar Sensitivity

2012 ◽  
Vol 78 (21) ◽  
pp. 7687-7697 ◽  
Author(s):  
F. Mark Dunning ◽  
Daniel R. Ruge ◽  
Timothy M. Piazza ◽  
Larry H. Stanker ◽  
Füsûn N. Zeytin ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
High Throughput ◽  
Botulinum Neurotoxin ◽  
Magnetic Beads ◽  
Mouse Bioassay ◽  
Current Standard ◽  
Complex Matrices ◽  
Serotype A ◽  
Wide Range ◽  
Botulinum Neurotoxin Serotype A

ABSTRACTRapid, high-throughput assays that detect and quantify botulinum neurotoxin (BoNT) activity in diverse matrices are required for environmental, clinical, pharmaceutical, and food testing. The current standard, the mouse bioassay, is sensitive but is low in throughput and precision. In this study, we present three biochemical assays for the detection and quantification of BoNT serotype A, B, and F proteolytic activities in complex matrices that offer picomolar to femtomolar sensitivity with small assay volumes and total assay times of less than 24 h. These assays consist of magnetic beads conjugated with BoNT serotype-specific antibodies that are used to purify BoNT from complex matrices before the quantification of bound BoNT proteolytic activity using the previously described BoTest reporter substrates. The matrices tested include human serum, whole milk, carrot juice, and baby food, as well as buffers containing common pharmaceutical excipients. The limits of detection were below 1 pM for BoNT/A and BoNT/F and below 10 pM for BoNT/B in most tested matrices using 200-μl samples and as low as 10 fM for BoNT/A with an increased sample volume. Together, these data describe rapid, robust, and high-throughput assays for BoNT detection that are compatible with a wide range of matrices.

scholarly journals Drosophila laminin: characterization and localization.

1987 ◽  
Vol 105 (5) ◽  
pp. 2383-2391 ◽  
Author(s):  
L I Fessler ◽  
A G Campbell ◽  
K G Duncan ◽  
J H Fessler
Keyword(s):  
Basement Membrane ◽  
Polyclonal Antibodies ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Basement Membranes ◽  
Velocity Sedimentation ◽  
Internal Organs ◽  
Differentiated Cells ◽  
Basement Membrane Component ◽  
Molecular Masses

Drosophila laminin was isolated from the medium of Drosophila Kc cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given. Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.

scholarly journals Can Thymidine Kinase 1 Detect Small Invisible Malignant Tumours?

2021 ◽  
Author(s):  
Ji Zhou ◽  
Huijun Li ◽  
Cong Fang ◽  
Junye Tan ◽  
Peng Gao ◽  
...  
Keyword(s):  
Thymidine Kinase ◽  
Total Protein ◽  
Magnetic Beads ◽  
Polyclonal Antibodies ◽  
Malignant Tumour ◽  
Malignant Cell ◽  
Cell Diameter ◽  
Malignant Tumours ◽  
Thymidine Kinase 1 ◽  
Protein Cell

Abstract Objectives. Early detection of malignant tumour is a prerequisite for a successful treatment. Here we investigate if thymidine kinase 1 is more sensitive than imaging technology to discover small invisible malignant tumours.Material and Methods. The cellular concentration of TK1 was determined by an automatic chemiluminescence analyzer of magnetic particle immune sandwich minimum. The primary and secondary antibodies linked to the magnetic beads were chicken anti-human thymidine kinase 1 IgY-polyclonal antibodies (IgY). The minimum number of cells able to detect by the automatic chemiluminescence analyzer were determined based on the cellar TK1 concentration of low and high TK1 cell lines of known cell count.Results. The TK1 concentration of malignant cell was found to be 0.021 pg/cell. Assuming 200 pg of total protein/cell, TK1 corresponds to 0.01 % of the total protein/cell. The concentration of TK1 in human blood serum of malignant patients is in the range of 2-10 pmol/l (pM), corresponding to about 50 x106 growing cells that release TK1 into 5 litre blood. The limit visibility by imaging of a tumour is about 1 mm in diameter, corresponding to about 109cells of a cell diameter of 1µm. Conclusion. TK1 is more sensitive than imaging.

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