Background

Immunoglobulins (Ig) or antibodies (Ab) are glycoproteins found in serum and tissue fluids, which are produced in large amounts after contact with immunogenic foreign molecules. They are Y-shaped and consist of five classes or isotypes: IgG, IgM, IgA, IgE, and IgD. Immunoglobulin G (IgG) is a type of antibody normally found in blood and extracellular fluid that is predominant in adaptive immune responses. The IgG isotype is 75% of normal serum immunoglobulins (14 mg/mL) and is divided into four sub-classes, called IgG1, IgG2, IgG3, and IgG4, in the following proportion: 70%, 20%, 8%, and 2%, respectively. All antibodies have the same basic structure divided into variable and constant regions; the variable region is antigen specific and contains two fragment antigen binding (Fab) sites, while the constant region, also known as fragment crystallizable (Fc), is isotype specific and stimulates antigen destruction. Human-derived IgGs are a subgroup of immunoglobulins that are commonly used for a variety of research methods or as effective therapeutics in inflammation, cancer, as well as autoimmune and infectious diseases. For in vitro methods, antibodies can be used directly in their crude form (serum) or in a pure form (IgG); however, purified antibodies are more advantageous than the whole serum because they reduce cross-reactivity, can be easily stored, and are stable for longer periods. Several autoimmune syndromes have been successfully "transferred" in animals immunized with highly purified human autoantibodies (Cuhadar et al., 2019; Goebel et al., 2021; Helyes et al., 2019; Pohoczky et al., 2022). Several protocols for antibody purification are available in the literature, and these are divided in two main groups: non chromatographic techniques, such as polyethylene glycol, and caprylic acid ammonium sulphate precipitation, and chromatographic techniques, such as affinity chromatography with protein A/G beads. Non-chromatographic techniques depend on the combination of several factors, and are therefore complex, laborious, and expensive, especially due to the usage of polyethylene glycol. Affinity chromatography with commercially available protein-A or protein-G beads is the standard methodology for purification of antibodies from serum, for the relative simplicity and reliability of results. While protein A has a higher affinity for rabbit, pig, dog, and cat IgG1, IgG2, and IgG4, protein G has a higher affinity for mouse and human IgG1, IgG2, IgG3, and IgG4. Furthermore, protein G beads have a wider binding range than protein A beads because they can bind to intact IgG as well as antibody fragments F(ab) 2 and Fc regions, while protein A binds strongly to the Fc part and weakly to the Fab region. Here, we provide a detailed and easy to follow protocol for purification of high-quality IgG from human serum, using affinity chromatography with protein G. Differently from previously published methods, this protocol is specifically designed for purification of large amounts of human serum (20 mL), whereby serum is diluted before purification, and Hartmann’s solution is used as a binding buffer instead of PBS, to keep neutral pH and physiological ionic strength(Cuhadar et al., 2019; Goebel et al., 2021; Helyes et al., 2019; Pohoczky et al., 2022). These steps significantly improved the human IgG purification. Furthermore, this protocol contains useful comments regarding factors affecting IgG binding and stability. This protocol could also be used for purification of antibodies from hybridoma culture supernatant or ascites.