Preparation of Polyclonal Antibody against B-cell activating factor

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Mardani Jouneghani Rasoul; Irani Shiva; Habibi Anbouhi Mahdi; Behdani Mahdi

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Journal Paper

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Journal: Iranian Journal of Physiology and Pharmacology Year:2023 | Volume:7 | Issue:1 Page(s): 197-205

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Title

Preparation of Polyclonal Antibody against B-cell activating factor

Author(s)

Mardani Jouneghani Rasoul | Irani Shiva | Habibi Anbouhi Mahdi | Behdani Mahdi | Issue Writer Certificate

Keywords

B-cell activating factor

Camel

Heavy chain antibody

Polyclonal antibody

Abstract

Background and Aim: B-cell activating factor (BAFF) belongs to the tumor necrosis factor (TNF) superfamily and plays a critical role in B-cell survival and differentiation. Overexpression of BAFF has been linked to autoimmune diseases and B-cell malignancies. The biologically active segment of this protein is a soluble domain, making it a promising target for antibody-based therapies. This study aimed to develop a polyclonal Camel antibody capable of detecting BAFF on cell surfaces. Methods: An expression vector, pET22b, containing the extracellular domain of the BAFF protein (NM_001145645.2), was constructed and introduced into Escherichia coli BL21 (DE3) using the calcium chloride heat shock method (CaCl2). Subsequently, the recombinant protein was induced using 1mM IPTG and protein purification was carried out with Ni2+–NTA resin. A 9-month-old female Camelus dromedaries was immunized with purified recombinant BAFF protein (rBAFF) mixed with Freund's adjuvant. Following immunization, the isolated Polyclonal antibody was assessed using ELISA, western blotting, and flow cytometry to detect rBAFF protein. Results: The study confirmed the successful preparation of recombinant BAFF protein and the effectiveness of Camel immunization and purification processes. The isolated Polyclonal antibody demonstrated the ability to identify recombinant BAFF proteins in ELISA and western blot tests. Flow cytometry demonstrated its capacity to detect BAFF protein on cell surfaces. Conclusion: Given the significance of diagnosing and developing novel adjuvant treatment methods for autoimmune diseases and cancer, this study's findings support the potential use of polyclonal antibodies targeting the BAFF antigen as valuable tools for these purposes.

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