RABBIT ANTI-MOUSE SERUM ALBUMIN POLYCLONAL ANTIBODY GENERATION FOR RELEASE TESTING OF MONOCLONAL ANTIBODIES PRODUCED FROM MURINE ASCITES FLUID

Authors

  • Isi Veitia
  • Jose Miguel Fernandez Center for Genetic Engineering and Biotechnology https://orcid.org/0000-0003-3832-509X
  • Ronny Camacho
  • Yeleiny Machín
  • Yaramis Luna
  • Liudmila Benítez
  • Duniesky Martínez
  • Joel Javier Pérez
  • Ricardo Pina
  • Lisbet Ulloa
  • Enrique Pérez
  • Yeosvany Cabrera

DOI:

https://doi.org/10.15414/jmbfs.5200

Keywords:

mouse serum albumin, polyclonal antibody, detection, ELISA, Western Blot

Abstract

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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Published

2021-11-24

How to Cite

Veitia, I. ., Fernandez, J. M., Camacho, R. ., Machín, Y. ., Luna, Y. ., Benítez, L. ., Martínez, D. ., Pérez, J. J. ., Pina, R. ., Ulloa, L. ., Pérez, E. ., & Cabrera, Y. . (2021). RABBIT ANTI-MOUSE SERUM ALBUMIN POLYCLONAL ANTIBODY GENERATION FOR RELEASE TESTING OF MONOCLONAL ANTIBODIES PRODUCED FROM MURINE ASCITES FLUID. Journal of Microbiology, Biotechnology and Food Sciences, e5200. https://doi.org/10.15414/jmbfs.5200

Issue

Section

Biotechnology
Received 2021-08-20
Accepted 2021-11-08
Online Published 2021-11-24