Regardless the technology applied for the evaluation of these conjugates, the need for better quality control tools raises as their application in different processes increases. Therefore, these conjugates can compromise the results from flow cytometry as well as from other technologies. Since the latest depends on the sort of energy excitation, which in a spectrometer is not present, one might have a good F/P ratio for a molecule, but not necessarily a satisfactory emission of the same molecule when it is tested in a flow cytometer. , the F/ P ratio does not necessarily express the fluorescence emission. After the conjugation process, the conjugates have to achieve their ideal F/P ratio determined in the conjugation protocol, which varies depending on the fluorochrome that is used. This ratio is determined by reading the optical densities (OD) of the antibody and fluorochromes in the spectrophotometer. The quality control of fluorescent conjugates is usually performed by spectrophotometry, where the ratio between fluorochrome and protein (F/P ratio) is measured. Hence, the credibility of the results obtained in this type of assays strongly depends on the conjugates performance. Those kits are applied to detect several types of molecules, such as drugs, hormones, infectious disease biomarkers and other types of antigens on antibody-based multiplex, enzyme-linked immunosorbent (ELISA) or flow cytometry assays. phycoerythrin-PE or fluorescein isothiocyanate-FITC), that make the reaction detectable. The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules, such as chromophores or fluorochromes (i.e. In therapy, there are numerous monoclonal antibodies licensed for the treatment of various diseases, like cancer, allergy and autoimmune diseases. In basic research, they are primarily used for staining both surface and intracellular proteins, like membrane receptors and cytokines. Monoclonal antibodies are glycoproteins containing uniform variable regions that confer a high specificity for a single epitope, favoring their use not only in scientific research, but also in immunodiagnostic and therapy. Our data demonstrated the feasibility of the flow cytometric method as a standard quality control of immunoassay kits. 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. Coefficients of variation (CVs) showed that this parameter can be used to determine better coupling conditions, such as homogenous coupling. The differences were observed between manufactures and lots from both anti-IgG-PE and anti-HBSAg-PE conjugates. Our results showed that there was a great difference in the fluorescence intensities between the conjugates studied. Their fluorescence intensities were followed for a period of 18 months. 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. 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. 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. The use of antibodies in immunodiagnostic kits generally implies the conjugation of these proteins with other molecules such as chromophores or fluorochromes.
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