Reevaluation of flow cytometry for investigating antibody binding to the surface of Plasmodium falciparum trophozoite-infected red blood cells.
Cytometry A
; 56(2): 96-103, 2003 Dec.
Article
en En
| MEDLINE
| ID: mdl-14608637
ABSTRACT
BACKGROUND:
The acquisition of antibodies directed toward variant surface antigens (VSAs) expressed on the surface of the trophozoite-infected red blood cell is an important determinant of natural immunity to Plasmodium falciparum malaria. In recent years, flow cytometry has been used increasingly to investigate these responses, but few systematic assessments of this method are available in the published literature.METHODS:
We developed a highly standardized experimental protocol and used parasites of the A4 laboratory clone, a monoclonal antibody to the VSA expressed by this clone (monoclonal antibody BC6), and a single pool of hyperimmune plasma to explore the parameters responsible for variations in VSA antibody responses measured by flow cytometry.RESULTS:
Despite strenuous efforts to standardize our flow cytometric assay, we found marked variability in our assay readout, even between repeat experiments using identical antibody and parasite combinations. We found no remediable cause for much of this variability. However, we identified three major factors that we considered important contributors antibody concentration, nonspecific antibody binding to uninfected red blood cells, and parasite agglutination.CONCLUSIONS:
A number of potential pitfalls should be considered when designing and interpreting studies using this technique. In particular, we suggest that comparisons between assays conducted on different occasions can be made only through reference to carefully selected standards. We anticipate that a better appreciation of the factors that lead to assay variation will assist the design of improved experimental protocols.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Plasmodium falciparum
/
Sitios de Unión de Anticuerpos
/
Anticuerpos Antiprotozoarios
/
Eritrocitos
/
Citometría de Flujo
/
Antígenos de Protozoos
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Cytometry A
Año:
2003
Tipo del documento:
Article
País de afiliación:
Kenia