Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies

Abstract

Background: Antibodies are the main effectors against malaria blood-stage parasites. Evaluation of functional activities in immune sera from Phase 2a/b vaccine trials may provide invaluable information in the search for immune correlates of protection. However, the presence of anti-malarial-drugs, improper collection/storage conditions or concomitant immune responses against other pathogens can contribute to non-specific anti-parasite activities when the sera/plasma are tested in vitro. Purification of immunoglobulin is a standard approach for reducing such non-specific background activities, but the purification method itself can alter the quality and yield of recovered Ag-specific antibodies.

Methods: To address this concern, various immunoglobulin (Ig) purification methods (protein G Sepharose, protein A/G Sepharose, polyethylene glycol and caprylic acid-ammonium sulphate precipitation) were evaluated for their impact on the quality, quantity and functional activity of purified rabbit and human Igs. The recovered Igs were analysed for yield and purity by SDS-PAGE, for quality by Ag-specific ELISAs (determining changes in titer, avidity and isotype distribution) and for functional activity by in vitro parasite growth inhibition assay (GIA).

Results: This comparison demonstrated that overall polyethylene glycol purification of human serum/plasma samples and protein G Sepharose purification of rabbit sera are optimal for recovering functional Ag-specific antibodies.

Conclusion: Consequently, critical consideration of the purification method is required to avoid selecting non-representative populations of recovered Ig, which could influence interpretations of vaccine efficacy, or affect the search for immune correlates of protection.

Figure 1

Procedure overview. Igs from either rabbit or human serum/plasma were purified (Step 1) and then biochemically characterized in (Step 2), and finally evaluated for the retention of biological activity (Step 3). Each method was tested in at least three independent experiments.

Figure 2

Ig purity depends on the species of the source material. MSP-1p42 specific titers of the Ig preparations, Panel A (rabbit samples), Panel B, C (human samples) were determined by ELISA and then adjusted to the pre-purification titer. All rabbit samples were diluted 1:50 and all human samples were diluted 1:10 to achieve optimal band resolution. Lanes: 1) molecular weight marker, 2) source material (pre-purification), 3) MW marker, 4) Protein G-purified Ig, 5) Protein A/G-purified Ig, 6) CA-AS-purified Ig and 7) PEG-purified Igs. Samples in Panel A, B were tested immediately following purification. Samples in Panel C were tested for their stability after storage for 24 weeks at 4°C. Panel C, 1) MW marker, 2) Protein G-purified Ig, 3) Protein A/G-purified Ig, 4) CA-AS-purified Ig and 5) PEG-purified Igs.

Figure 3

Changes in Ag-specific titers as a function of purification methods. Various Ig preparations from (A) rabbit sera or (B) human serum or plasma were tested for changes in the MSP-1p42 specific titer (i.e., OD = 1 at 405 nm). Data shown are the geometric mean and 95% confidence interval of three independent experiments for each purification method. Asterisks indicate statistically significant differences (p < 0.05).

Figure 4

Effect of purification methods on isotype subclass distribution in Ig preparations. Ig preparations purified using the various methods were tested for changes in MSP-1p42 specific IgG1 (Panel A), IgG2 (Panel B), IgG3 (Panel C) and IgG4 (Panel D) for each treatment; pre-treatment serum, Protein G, Protein A/G, CA-AS, and PEG. Immune serum was from a naturally exposed individual residing in Gabon. Data shown are the mean OD405 (± SEM) of three independent isotype subclass specific ELISA experiments. Asterisks indicate statistically significant differences (p < 0.05). Dashed lines indicate background level responses for each isotype subclass test.

Figure 5

Growth inhibitory activity of various Ig preparations from (A) MSP-1p42 (FVO) specific rabbit sera or (B) human malaria-experienced plasma was measured against FVO parasites in a pLDH based growth inhibition assay (GIA). Data are expressed as the mean and SEM of three independent purifications per method (sample size for all treatment groups n = 27 except for Protein A/G (n = 6)). Asterisk indicates statistically significant differences (p < 0.01).