Keywords
GAD65-specific, monoclonal autoantibody, affinity purification, autoantibody production
This article is included in the Antibody Validations gateway.
GAD65-specific, monoclonal autoantibody, affinity purification, autoantibody production
Type 1 diabetes (T1D) is an autoimmune disorder characterized by the immune-mediated destruction of the insulin-producing β cells in the pancreas. Human islet cells express the 65-kDa isoform of glutamic acid decarboxylase (GAD65), which is one of the most common autoantigens associated with the development of T1D. Anti-GAD65 autoantibodies (GAD65Abs) are detectable several years before diabetes and present in over 70% of patients at the time of diagnosis1. It has been suggested that healthy individuals also generate GAD65Abs, which are sufficiently neutralized by anti-idiotypic antibodies (anti-Id Abs), resulting in protection from GAD65-specific islet destruction2,3. Probably because the antigen-binding region of GAD65Abs is blocked by anti-Id Abs, circulating GAD65Abs in sera of healthy individuals are not detectable using GAD65-specific methods. The decline of anti-Id Abs in patients developing T1D, on the contrary, unmasks GAD65Abs, which then serve as critical serum markers in prediction and diagnostics of diabetes4. Studies of the interaction between GAD65 and recombinant GAD65Abs have suggested immunodominant epitopes on GAD655–9. However, how the recognition of these epitopes by GAD65Abs drives islet destruction, and how anti-Id Abs block GAD65Ab-mediated auto-reactivity are largely unknown. In order to generate anti-Id Abs aimed at understanding of pathophysiologic mechanism(s), and more importantly, preventing GAD65 autoreactivity, it is necessary to isolate and utilize native GAD65Abs rather than synthesizing recombinant proteins. However, no published data have ever reported on the quality of purified GAD65Abs for such aims, even though two of these human Abs (b96.11 and b78)10–13 are commercialized.
Certain limitations stem from technical issues in the purification and characterization of native GAD65Abs originated from T1D patients. The most efficient way to produce monoclonal autoantibodies in vitro is to generate monoclonal B cell lines, culture them in batches, and purify the Abs from the culture supernatant. Although many established methods have been standardized for Ab purification14, the polymorphic nature of Abs and the diverse culture conditions of Ab-secreting cell lines may impede the achievement of native autoantibody products with satisfactory quality and purity. Utilization of impure GAD65Abs in the generation of anti-Id Abs and determination of their protective role in T1D pathogenesis may lead to unconvincing or inconclusive results.
In this report, we evaluated multiple strategies for the purification of two human monoclonal GAD65Abs: DPA and DPD10. Our goal was to isolate a pure population of Abs with minimal contaminants. We also determined GAD65-binding affinity of these two autoantibodies as the initial step of molecular characterization.
Detailed information on reagents used in this study is listed in Table 1.
The monoclonal B cell lines secreting either DPA or DPD were immortalized by Epstein-Barr virus (EBV) transformation as described10. These cell lines were maintained in complete Iscove’s modified Dulbecco’s medium (IMDM); or adapted to serum-free medium by diluting at a ratio of 1:2–1:3 every three days followed by a complete replacement after 10 days. Five million live cells were pelleted and reverse transcriptase polymerase chain reaction (RT-PCR) performed with the SuperScript III First-Strand Synthesis System (Life Technology) and antibody-specific primers (Table 2).
* SN: sense primer. All SN primer sequences were described previously10.
The supernatants of cell cultures containing Abs were filtered through a 0.22 μm membrane to remove cell debris. Abs were purified from the supernatant by affinity chromatography (as per manufacturer’s instructions (Table 1)), followed by size exclusion chromatography (SEC) using a Superdex 200 gel filtration column (GE Healthcare). Fractions containing monomeric forms of each protein were pooled and analyzed by Coomassie stain or western blot.
Purified immunoglobulin G (IgG) products were reduced in sodium-dodecyl-sulphate (SDS) -containing Laemmli sample buffer with freshly added β-mercaptoethanol (βME) and denatured by boiling at 100°C for 10 min before separation by gel electrophoresis using Mini-PROTEAN TGX precast polyacrylamide gels (Bio-Rad). The gels were stained with SimplyBlue SafeStain (Life Technology) and destained with Milli-Q water for at least 1 h before imaging of IgG heavy and light chains. To differentiate the heavy and light chains of human IgG from non-specific contaminants co-purified from cell culture supernatant, proteins on the gel were transferred to Immobilon-P membrane (EMD Millipore) for human IgG detection. Goat F(ab’)2 anti-human Ig (2.5 mg/ml, Life Technology, Inc; used at 1:3000 dilution) followed by HRP-donkey anti-goat IgG (0.4 mg/ml, Santa Cruz Biotechnology, Inc; used at 1:10000 dilution) (see Table 3 for full Ab information) were used to detect human Ig.
Recombinant GAD65 (a gift from Peter van Endert, Institut National de la Santé et de la Recherche Médicale, France) in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)+1 mM pyridoxal phosphate (PLP)+50% Glycerol, pH 7.4, was stored in aliquots at -80°C; and the reactivity was verified by ELISA using commercially available mouse anti-GAD65 IgG1 and horseradish peroxidase (HRP) labeled goat anti-mouse IgG1 (Table 3). The ELISA protocol for GAD65/autoantibody interaction has been described (see Table 4).
Both GAD65Abs purified in this study belong to the human IgG1 (γ1) subclass; DPA uses a λ light chain and DPD uses a κ light chain10. Prior to the purification of soluble DPA or DPD IgG from cell culture supernatant, we first validated Ig cDNA expression in each cell line using standard RT-PCR (Figure 1). Note that we used an anti-sense oligonucleotide to prime the 3'-end of membrane IgG heavy chain cytoplasmic domain instead of one priming the 3'-end of the IgG heavy chain constant region used elsewhere, in order to generate the entire sequence of the heavy chain (Supplementary file S1).
Although both anti-GAD65 Ab-secreting cell lines are derived from peripheral blood mononuclear cells (PBMCs) of a T1D patient10, their culture conditions are significantly different. The DPA cell line expanded well in both serum-supplemented and serum-free medium, while the DPD cell line survived only in serum-supplemented medium. Fetal bovine serum (FBS) is widely used in tissue-culture medium to provide essential proteins, nutrients and other uncharacterized factors for optimum cell growth; however, the presence of bovine IgG (bIgG) in the serum (up to 50 mg/L) is the main source of contamination in human IgG (hIgG) purification. Bovine serum albumin (BSA) is also commonly used at a high concentration in culture medium (can be over 1 mg/ml), and binds non-specifically during the protein purification process.
Affinity purification using antigens or IgG-binding proteins (e.g., Protein A, G and L) is very effective for Ab production, with antigen affinity purification being the most specific technique and providing the purest batches of antibody. However, GAD65Ab purification using recombinant GAD65 (rGAD65) for antigen-specific affinity purification is difficult because rGAD65 is unstable and requires pyridoxal phosphate (PLP) for stabilization. Considering the inevitable exposure of rGAD65 pre-coupled to resin to the extreme pH (<4 or >10) in elution and regeneration steps, this would not be a viable option. We therefore chose IgG-binding proteins in our attempt to affinity purify GAD65Abs without potential protein contaminants. Both Protein A and G recognize the Fc domain of IgG from human and bovine sera, while protein L binds to κ light chain. Gammabind sepharose beads (GE healthcare) use a recombinant form of Protein G (rProtein G), which significantly reduces the non-specific binding of BSA to the resin. Purification of IgG from the supernatant of DPA cell culture (grown in FBS-containing medium) on rProtein G resin resulted in purer IgG (Figure 2A), than using native Protein A resin (nProtein A) (Figure 2B). However, the purified IgG products from both rProtein G and nProtein A still contained a high molecular-weight (MW; MW>100 kDa) component besides the anticipated heavy chain (~50 kDa) and light chain (25 kDa) on coomassie-stained protein gels. Western blotting analysis suggested that this component did not belong to human Ig (Figure 2C). The relative percentage of contamination with the high MW protein in IgG purified using nProtein A was significantly lower than when purified with rProtein G (Figure 2A, Figure 2B). This component may reflect bIgG-associated contaminants, as bIgG has lower binding affinity for nProtein A than rProtein G. To test this, we gradually adapted DPA cells from FBS-containing medium to FBS-free medium and were able to affinity-purify hIgG from the culture supernatant without bIgG using rProtein G (Figure 2D). We further separated DPA hIgG from any BSA contamination by SEC. The comparison between DPA purified using different methods and bIgG purified from pure FBS confirmed that the high MW contaminate is associated with bIgG (Figure 2D and Figure 3). Importantly, we demonstrated that serum-free culture is key to isolating highly pure DPA hIgG.
In contrast, DPD did not grow well in the serum-free medium we tested, and thus we opted to use Protein L as an alternative method to obtain more pure hIgG from this line. Protein L binds the light chain of IgG and DPD has a κ light chain. Notably, no previous evidence suggested that Protein L distinguishes κ chain of hIgG from bIgG; however, we found that Protein L affinity purification followed by SEC separation generated DPD hIgG with satisfactory purity and no detectable bIgG or bIgG-associated high MW proteins even though DPD cell culture contains 10% FBS (Figure 2E). We also demonstrated that ion-exchange chromatography is not appropriate to separate hIgG from bIgG, as the high MW bIgG-associated protein(s) were present in all fractions eluted from anion or cation exchange columns (Figure 4).
We then determined the binding affinity of the purified DPA and DPD to rGAD65 by ELISA (Figure 5). Given the instability of rGAD65, the measurement of its concentration was inaccurate. To overcome this problem, we coated the ELISA plate with two concentrations of rGAD65 (10–100 nM) differing by 3-fold and incubated immobilized rGAD65 with titrated amounts of purified DPA or DPD monoclonal Abs at 37°C for 2 h. The concentration of immobilized rGAD65 did not influence the calculation of the dissociation constant (KD). We assumed that the duration of incubation was sufficient for the interaction between rGAD65 and GAD65Ab to reach equilibrium and fitted the data to a single site binding equation:
y=Bmax*x/(KD+x)
to estimate KD (Table 5). Purified DPA has over 100-fold higher rGAD65-binding affinity (the inverse of KD) than purified DPD.
To the best of our knowledge, purification and characterization of native GAD65Ab, free from culture medium-derived contaminants such as bIgG and BSA, have not been reported previously, in spite of the availability of monoclonal cell lines secreting these Abs10,11. Our goal was to obtain a very pure preparation of GAD65-specific hIgG. Here, we demonstrate several strategies to overcome limitations associated with affinity purification that would be applicable to the purification of many other antibodies: (1) antigen-specific affinity purification is always superior, if the autoantigen itself can be easily produced and can tolerate exposure to pH extremes; (2) when dealing with an unstable autoantigen (most often), the attempt to adapt cells to serum-free medium is worthwhile to avoid bIgG contamination; (3) Protein L recognizes the light chains of Ig from different species, however, as we have shown here Protein L may preferentially bind human rather than bovine κ chain and provide an alternative approach to purification of autoreactive hIgG(κ).
It is of interest that there is over 100-fold difference in the rGAD65-binding affinity between DPA and DPD. Without understanding the mechanism, it is hard to predict the relationship between autoantigen binding affinity and the severity of disease. However, this finding reminds us that low affinity autoantibodies indeed exist, but are less likely to be detected in diagnostic tests, considering the binding of GAD65Abs by anti-Id Abs. Therefore, the detection threshold in diagnostic tests for measuring GAD65Abs or other autoantibodies in patient sera may need further optimization for a more thorough monitoring of low affinity autoantibodies and prediction of T1D.
WJ and EDM conceived the study. WJ designed the experiment, carried out the research and prepared the first draft of the manuscript. HM contributed to the design of experiments and provided expertise in cell culture. AM provided the DPA and DPD cell lines. All authors were involved in the preparation and revision of the draft manuscript and have agreed to the final content.
This work was supported by National Institutes of Health Grants AI095813 and AI075253 (both to E.D.M.), the Stanford NIH/NCRR CTSA award UL1 RR025744 (to W.J.), National Institutes of Health/National Center for Research Resources Clinical and Translational Science Award UL1 RR025744 (to H.M.), and the Lucile Packard Foundation for Children’s Health. H.M. was also supported by National Institutes of Health Training Grant T32 AI07290 to the Stanford Interdisciplinary Program in Immunology.
I confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We thank Peter van Endert (Institut National de la Santé et de la Recherche Médicale, France) for providing the recombinant GAD65 protein and Taejin Yoon (former member of the Mellins laboratory) for providing advice and expertise in protein purification.
Views | Downloads | |
---|---|---|
F1000Research | - | - |
PubMed Central
Data from PMC are received and updated monthly.
|
- | - |
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | |||
---|---|---|---|
1 | 2 | 3 | |
Version 2 (revision) 21 Apr 16 |
read | ||
Version 1 29 May 15 |
read | read |
Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list:
Sign up for content alerts and receive a weekly or monthly email with all newly published articles
Already registered? Sign in
The email address should be the one you originally registered with F1000.
You registered with F1000 via Google, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Google account password, please click here.
You registered with F1000 via Facebook, so we cannot reset your password.
To sign in, please click here.
If you still need help with your Facebook account password, please click here.
If your email address is registered with us, we will email you instructions to reset your password.
If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance.
Comments on this article Comments (0)