Quantitation of complement and leukocyte binding to a parasitic helminth species.
Methods used to quantify complement deposition and cell adherence to parasitic helminths usually involve subjective visual comparisons of immunofluorescence or time-consuming manual counting of bound cells. Such targets are relatively large and, generally, few individual organisms can be analysed. More objective and efficient radiometric assays are available, but these also have considerable disadvantages. We have developed an improved immunofluorescence-based method for quantitation of complement deposition on viable third-stage larvae of the nematode Nippostrongylus brasiliensis (L3). A similar strategy was also applied to measuring leukocyte adherence to the parasite. Fluorescein isothiocyanate (FITC)-conjugated antibodies were used to detect complement on serum-treated larvae. The adherence of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled mouse leukocytes to larvae was investigated using the same basic approach. Images of fluorescent larvae or fluorescent cells attached to larvae were generated with a Bio-Rad Molecular Imager FX and fluorescence intensity was quantified. Hundreds of larvae can be analysed simultaneously in multiple samples, and these strategies allow rapid and sensitive quantitation that is directly proportional to the amount of protein or the number of leukocytes added to cultures. These techniques may also be applicable to other large objects, organisms or biological surfaces.
Giacomin
Paul R
PR
School of Molecular and Biomedical Science, University of Adelaide, North Tce, South Australia, Australia.
Wang
Hui
H
Gordon
David L
DL
Dent
Lindsay A
LA
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002448:Cell Adhesion / Q000276:immunology; D003176:Complement C3 / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007814:Larva / Q000276:immunology; D007962:Leukocytes / Q000737:chemistry / Q000276:immunology* / Q000469:parasitology*; D051379:Mice; D008808:Mice, Inbred CBA; D009559:Nippostrongylus / Q000276:immunology*; D044967:Serum / Q000276:immunology; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000906:Antibodies; D003176:Complement C3; D005452:Fluoresceins; D013388:Succinimides; D016650:Fluorescein-5-isothiocyanate