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.

Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease

“…The development of this technique has been possible by using samples from non‐SCD subjects presenting a homogeneous distribution of HbF for whom the mean HbF fluorescence can be directly related to the MCHbF. Despite the choice of an accurate monoclonal antibody, the slight variability we observed came from the batch 26 . The integration of the variability of the degree of coupling of PE molecules per antibody, by applying a correction factor to the measured fluorescence intensities, allows greater accuracy of the assay with good reproducibility and repeatability.…”