A comparison between different chemical fractionation methods for immunoglobulin preparation.

Q2 Health Professions Journal of immunoassay & immunochemistry Pub Date : 2025-01-11 DOI:10.1080/15321819.2025.2450664

Abbasali Salarifar, Pooria Safarzadeh Kozani, Mohammad Javad Rasaee

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Abstract

Background: Application of antibodies in therapeutics and diagnostics are growing Continually. Herein, we aimed to find the most qualified immunoglobulin (Ig) chemical preparation method.

Methods: A rabbit was immunized against recombinant SARS-CoV-2 nucleocapsid (NP) and reactive polyclonal antibodies were prepared using the ammonium sulfate (AS), caprylic acid (CA), polyethylene glycol (PEG), and caprylic acid/ammonium sulfate (CA/AS) methods. Different antibody solutions were analyzed by SDS-PAGE and subsequently quantified by ImageJ software for further analysis in terms of Ig purity, Ig recovery, and albumin impurity. Ultimately, the prepared antibodies were assessed via Western blotting and ELISA to evaluate their ability to bind NP.

Results: Prepared Ig solutions via the CA/AS method had the highest Ig purity (followed by CA, PEG, and AS) and lowest albumin impurity (followed by CA, AS, and PEG). The PEG method had the highest recovery followed by AS, CA, and CA/AS methods. Moreover, antibodies prepared via different methods demonstrated comparable binding capacities to NP in ELISA and Western blotting.

Conclusions: CA/AS, closely followed by CA, proved to be the most qualified method for the preparation of Ig yielding the highest Ig purity while the PEG method resulted in the highest Ig recovery rate.

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制备免疫球蛋白不同化学分馏方法的比较。

背景：抗体在治疗和诊断中的应用不断增长。在此，我们旨在寻找最合格的免疫球蛋白（Ig）化学制备方法。方法：采用硫酸铵（AS）、辛酸（CA）、聚乙二醇（PEG）和辛酸/硫酸铵（CA/AS）法制备重组SARS-CoV-2核衣壳（NP）免疫兔，制备反应性多克隆抗体。通过SDS-PAGE分析不同抗体溶液，然后通过ImageJ软件进行定量分析，进一步分析Ig纯度、Ig回收率和白蛋白杂质。最后，通过Western blotting和ELISA评估制备的抗体与NP的结合能力。结果：通过CA/AS法制备的Ig溶液中，Ig纯度最高（其次是CA、PEG和AS），白蛋白杂质最低（其次是CA、AS和PEG）。PEG法回收率最高，其次为AS法、CA法和CA/AS法。此外，通过不同方法制备的抗体在ELISA和Western blotting中显示出与NP相似的结合能力。结论：CA/AS法制备Ig纯度最高，其次是CA法，PEG法提取Ig回收率最高。

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来源期刊

Journal of immunoassay & immunochemistry 医学-免疫学

CiteScore

3.50

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： The Journal of Immunoassay & Immunochemistry is an international forum for rapid dissemination of research results and methodologies dealing with all aspects of immunoassay and immunochemistry, as well as selected aspects of immunology. They include receptor assay, enzyme-linked immunosorbent assay (ELISA) in all of its embodiments, ligand-based assays, biological markers of ligand-receptor interaction, in vivo and in vitro diagnostic reagents and techniques, diagnosis of AIDS, point-of-care testing, clinical immunology, antibody isolation and purification, and others.