Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Oct;425:27-36.
doi: 10.1016/j.jim.2015.06.005. Epub 2015 Jun 12.

Microscale Purification of Antigen-Specific Antibodies

Affiliations
Free PMC article

Microscale Purification of Antigen-Specific Antibodies

Eric P Brown et al. J Immunol Methods. .
Free PMC article

Abstract

Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain.

Keywords: Antibody; Antigen; Effector function; Glycosylation; Purification.

Figures

Fig. 1
Fig. 1
Assay schematic. A) Polyclonal antibodies present in either purified IgG or serum are bound to antigen-coated cartridges and washed to remove all non-specific IgG. Antigen-specific antibodies can then be eluted with either low pH citrate buffer, which releases the full Ig, or an IdeS enzyme solution, which cleaves and releases the Fc portion allowing separation of Fab′2 and Fc domains. Full Ig, cleaved Fc, or cleaved Fab′2 can then be used for glycan analysis. B) Schematic of IdeS cleavage resulting in unbound Fc fragments.
Fig. 2
Fig. 2
Purification of diverse specificities, reproducibility and quality of purified antibody. (A–E) Antibodies specific for HIV gp120 (A), gp41 (B), p24 (C), an SIV V2 loop peptide (D), and the influenza HA protein (E) were purified from HIVIG (A/B/C), IVIG (E), or an SIV-vaccinated macaque (D). The ability of the antibody load (Input), flowthrough (FT), and the eluted antibody fraction (Elution) to bind to each antigen of interest across a range of sample concentrations was measured via an antigen-specific multiplex assay. The concentration versus binding signal of the gp120-specific monoclonal antibody b12 is shown as a benchmark of sample purity (A). Results are representative of at least 3 replicates. (F–H) HIV gp120-specific Abs were purified from HIVIG in quadruplicate. Yield (F) and ability to bind gp120, p24 and p17 were evaluated for each fraction of each purification replicate (G). Extended binding curves were used to calculate the reciprocal EC50s of each sample relative to the average observed across the loaded antibody sample toward each antigen (H).
Fig. 3
Fig. 3
Yield and enrichment factors. A) The yield of HIV gp120-specific antibodies isolated from a set of 44 HIV-positive subjects and SIV gp120-specific antibodies from 60 vaccinated rhesus macaques is presented. B) Antigen (gp120) binding titration curves of loaded antibody samples (serum) and eluted fractions (eluate) from a representative subset of the rhesus purifications (n = 7) from part A are plotted. C) The fold-enrichment of antigen specific antibodies as determined by calculation of the minimal antibody concentration at which binding signals 3× above background were observed for loaded and eluted serum samples described in B.
Fig. 4
Fig. 4
Glycosylation profiles of multiple antigen-specificities from a single source. A) Representative HPLC glycan data, demonstrating the overlay of chromatography traces for bulk HIVIG glycoforms (red) and gp120-specific antibodies purified from HIVIG (green) with peak identities and representative structural cartoons. B) Relative abundance of glycoforms present in HIVIG, and among gp120, gp41, and p24-specific antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Fc glycan analysis of IdeS-eluted antigen specific antibodies. Antibodies specific to HIV gp120 were purified from four chronically infected HIV patients, and Fc domains released using an on-cartridge IdeS elution. These samples, and Fc domain isolated from total plasma IgG (bulk) were profiled via capillary electrophoresis for glycan composition. A–F) The relative prevalences of several major classes of glycoforms are presented for each matched gp120 and bulk Fc sample. G–I) The relative prevalences of all identifiable glycan species (G), differentially galactosylated (H), and sialylated (I) forms averaged across subjects. A paired Student's t-test was conducted to determine the significance of differences observed in glycan prevalences between specificities (*p < 0.05, **p < .005).

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback