title: Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays.
abstract: The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules such as chromophores or fluorochromes. The development of more sensitive quality control procedures than spectrophotometry is essential to assure the use of better fluorescent conjugates since the fluorescent conjugates are critical reagents for a variety of immunodiagnostic kits. In this article, we demonstrate a new flow cytometric protocol to evaluate conjugates by molecules of equivalent soluble fluorochromes (MESF) and by traditional flow cytometric analysis. We have coupled microspheres with anti-IgG-PE and anti-HBSAg-PE conjugates from distinct manufactures and/or different lots and evaluated by flow cytometry. Their fluorescence intensities were followed for a period of 18 months. Our results showed that there was a great difference in the fluorescence intensities between the conjugates studied. The differences were observed between manufactures and lots from both anti-IgG-PE and anti-HBSAg-PE conjugates. Coefficients of variation (CVs) showed that this parameter can be used to determine better coupling conditions, such as homogenous coupling. The MESF analysis, as well as geometric mean evaluation by traditional flow cytometry, showed a decrease in the values for all conjugates during the study and were indispensable tools to validate the results of stability tests. Our data demonstrated the feasibility of the flow cytometric method as a standard quality control of immunoassay kits.
authors.affiliation: Laboratory of Diagnostic Technology, Immunobiological Technology Institute, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Marta
authors.initials: M
authors.lastname: de Almeida Santiago
authors.affiliation: Laboratory of Diagnostic Technology, Immunobiological Technology Institute, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Bruna
authors.initials: B
authors.lastname: de Paula Fonseca E Fonseca
authors.affiliation: Laboratory of Diagnostic Technology, Immunobiological Technology Institute, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Christiane de Fátima
authors.initials: CF
authors.lastname: da Silva Marques
authors.affiliation: Laboratory of Diagnostic Technology, Immunobiological Technology Institute, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Edimilson
authors.initials: E
authors.lastname: Domingos da Silva
authors.affiliation: Laboratory of Immunoparasitology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil.|Flow Cytometry Core Facility, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Alvaro Luiz
authors.initials: AL
authors.lastname: Bertho
authors.affiliation: Laboratory of Clinical Immunology, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana Cristina Martins de Almeida
authors.initials: AC
authors.lastname: Nogueira
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000276:immunology; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D018796:Immunoconjugates / Q000737:chemistry* / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008863:Microspheres; D010799:Phycoerythrin / Q000737:chemistry; D011786:Quality Control
chemical_list: D000888:Antibodies, Anti-Idiotypic; D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D006514:Hepatitis B Surface Antigens; D018796:Immunoconjugates; D007074:Immunoglobulin G; C048844:anti-IgG; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate