RABBIT ANTI-MOUSE SERUM ALBUMIN POLYCLONAL ANTIBODY GENERATION FOR RELEASE TESTING OF MONOCLONAL ANTIBODIES PRODUCED FROM MURINE ASCITES FLUID
Keywords:mouse serum albumin, polyclonal antibody, detection, ELISA, Western Blot
Monoclonal antibodies (mAbs) have become essential analytical tools for biomedical and veterinary diagnosis and the analytical release of other biomolecules due to their high specificity and reproducibility. The mAbs generated in mice contain murine serum albumin (MSA) as the main potential contaminant; this molecule interferes in a large number of tests in which these mAbs are required. For this reason, the development of analytical methods to identify and quantify MSA traces plays an important role in the quality of purified mAb. In this study, an anti-MSA polyclonal antibody (pAb) was generated in rabbit with titers of 1:2 700 000 and purified by nProtein A affinity chromatography with purity greater than 90%. The purified antibody was conjugated to horseradish peroxidase (HRP) by the m-periodate method with an optimal working dilution of 1:64 000 in direct ELISA. Two applications were evaluated for the analytical release of mAbs: identity test by Western Blot and direct ELISA, and albumin quantification by sandwich ELISA. The pAb showed a high specificity capable of identifying MSA traces in the formulations evaluated. The sandwich ELISA demonstrated the ability to quantify MSA in 0.49-125 ng/mL range. These assays are suitable for screening multiple samples, allow rigorous analytical release of mAbs, and assess batch-to-batch consistency.
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Copyright (c) 2022 Isi Veitia, Jose Miguel Fernandez, Ronny Camacho, Yeleiny MachÃn, Yaramis Luna, Liudmila BenÃtez, Duniesky MartÃnez, Joel Javier PÃ©rez, Ricardo Pina, Lisbet Ulloa, Enrique PÃ©rez, Yeosvany Cabrera
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Online Published 2022-04-01