Immunocapture of circulating Schistosoma mansoni cathepsin B antigen (Sm31) by anti-Sm31 polyclonal antibodies.

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Adult Schistosoma mansoni parasites have the capacity to degrade ingested host hemoglobin and other host plasma proteins by using a series of gut proteolytic enzymes, including cathepsin B; this enzyme is released to the host intravascular environment during regurgitations of adult worms.
Cathepsin B becomes thus a circulating parasite component that has been shown to be specifically recognized as the Sm31 antigen by antibodies present in most S. mansoni infected patients.
Taking advantage of this immunological property, we attempted here to immunocapture Sm31 from sera of infected patients using specific polyclonal rabbit antibodies raised against a highly enriched preparation of Sm31 and detect its intrinsic proteolytic activity using a previously described solid-phase procedure called Cysteine Protease Immuno Assay (CPIA).
To produce highly specific anti-Sm31/cathepsin B antibodies, cathepsin B (Sm31 or SmCB) was enriched more than 3000-folds from an adult worm preparation using a series of conventional biochemical steps including ion exchange and affinity chromatography.
Anti-cathepsin B antibodies were generated by immunizing rabbits with the enriched cathepsin B fraction; these antibodies recognized a band of Mr.~31 kDa in Western-blot (WB) analysis of this fraction and were able to capture, in a modified CPIA procedure, Sm31/SmCB present in sera from infected Venezuelan patients living in low endemic areas for schistosomiasis.
CPIA showed 100% sensitivity and 100% specificity; representing a new diagnostic tool to detect circulating Sm31 antigen in actual infections.

Life-threatening hemolytic anemia due to an autoanti-Pr cold agglutinin: evidence that glycophorin A antibodies may induce lipid bilayer exposure and cation permeability independent of agglutination

BACKGROUND
The hemoglobin of a 29-year-old man fell below 35 g/L over 5 days, despite 14 units of red blood cells (RBCs), due to an anti-Pr cold agglutinin (CA).
His hemolytic anemia necessitated respiratory support in intensive care for 4 weeks.
METHODS
The hemolysis was investigated by the effects on blood group-compatible RBCs of this anti-Pr and an anti-I CA and of a rabbit anti-human glycophorin A (GPA) immunoglobulin G (IgG) antibody on Ca(2+) permeability and of phosphatidylethanolamine (PE) exposure.
1) The anti-Pr CA (in a plasmapheresis product from the patient) was absorbed and eluted from RBC ghosts and its immunophenotype was determined by agarose electrophoresis and immunofixation.
2) Ca(2+) permeability was measured by the response of Fluo-3-labeled RBCs to addition of external Ca(2+).
3) Exposed PE was measured with streptavidin-labeled biotinylated peptide Ro 09-0198 (cinnamycin).
RESULTS
1) The patient’s anti-Pr CA was a polyclonal IgG.
2) The anti-Pr and the rabbit anti-human glycophorin IgG, but not an anti-I CA, rapidly increased Ca(2+)-dependent fluorescence upon addition of external Ca(2+) in a fraction (15%-25%) of RBCs that also became positive for cinnamycin.
3) Trypsin treatment of RBCs reduced the Ca(2+) influx due to the anti-Pr IgG, but neither trypsin nor neuraminidase changed the responses to the rabbit anti-human GPA IgG.
CONCLUSIONS
The anti-Pr CA and rabbit anti-human GPA increased exposure of PE and increased membrane Ca(2+) permeability that may have caused hemolysis.
The difference in the responses to these antibodies to enzyme treatment of RBCs suggests that they react with different epitopes on GPA.

Development of enantioselective polyclonal antibodies to detect styrene oxide protein adducts.

Styrene has been reported to be pneumotoxic and hepatotoxic in humans and animals. Styrene oxide, a major reactive metabolite of styrene, has been found to form covalent binding with proteins, such as albumin and hemoglobin.
Styrene oxide has two optical isomers and it was reported that the (R)-enantiomer was more toxic than the (S)-enantiomer.
The purpose of this study was to develop polyclonal antibodies that can stereoselectively recognize proteins modified by styrene oxide enantiomers at cysteine residues. Immunogens were prepared by alkylation of thiolated keyhole limpet hemocyanin (KLH) with styrene oxide enantiomers.
Polyclonal antibodies were raised by immunization of rabbits with the chiral immunogens.
Titration tests showed all six rabbits generated high titers of antisera that recognize (R)- or (S)-coating antigens accordingly.
No cross-reaction was observed toward the carrier protein (BSA). All three rabbits immunized with (R)-immunogen produced antibodies that show enantioselectivity to the corresponding antigen, while only one among the three rabbits immunized with (S)-immunogen generated antibodies with enantioselectivity of the recognition.
The enantioselectivity was also observed in competitive ELISA and immunoblot analysis.
Additionally, competitive ELISA tests showed that the immunorecognition required the hydroxyl group of the haptens.
Immunoblot analysis demonstrated that the immunorecognition depended on the amount of protein adducts blotted and hapten loading in protein adducts.
In summary, we successfully developed polyclonal antibodies to stereoselectively detect protein adducts modified by styrene oxide enantiomers.

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