Article
Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays
Registro en:
SANTIAGO, Marta de Almeida: et al. Flow Cytometry as a Tool for Quality Control of Fluorescent Conjugates Used in Immunoassays. Plos One, v.11, n.12, e0167669, Nov. 2016.
1932-6203
10.1371/journal.pone.0167669
Autor
Santiago, Marta de Almeida
Fonseca, Bruna de Paula Fonseca e
Marques, Christiane de Fátima da Silva
Silva, Edimilson Domingos da
Bertho, Álvaro Luiz
Nogueira, Ana Cristina Martins de Almeida
Resumen
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.