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ISSN: 2161-1009
Biochemistry & Analytical Biochemistry
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Overview of IgG-Reactivity in Therapeutic immunoglobulins Revealed by Protein Array Analysis

Sonia Néron1,2* and Annie Roy1
1Héma-Québec, Ingénierie cellulaire, Recherche et Développement, 1070 avenue des Sciences-de-la-vie, Québec, Québec, Canada, G1V 5C3
2Université Laval, Faculté des sciences et de génie, Département de Biochimie et Microbiologie, 1045 avenue de la Médecine, Québec, Québec,Canada, G1V 0A6
Corresponding Author : Sonia Néron
Héma-Québec, Recherche et Développement
1070 avenue des Sciences-de-la-vie
Québec (Québec), Canada, G1V 5C3
Tel:1 (418) 780-4362 (ext. 3260)
Fax: 1 (418) 780-2091
E-mail: [email protected]
Received May 17, 2012; Accepted July 23, 2012; Published July 26, 2012
Citation: Néron S, Roy A (2012) Overview of IgG-Reactivity in Therapeutic immunoglobulins Revealed by Protein Array Analysis. Biochem Anal Biochem S8:001. doi: 10.4172/2161-1009.S8-001
 
Copyright: ©2012 Néron S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 
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Abstract

In addition to therapy for immune deficient patients, intravenous immune globulins (IVIg) are used to treat several autoimmune and inflammatory diseases. Several groups working to better understand how IVIg can modulate immune and inflammatory responses have proposed the involvement of self-reactive IgG present in IVIg. Consequently, a moreexhaustive characterization of IVIg repertoire could be very helpful for their better utilization. To increase our knowledge on IVIg repertoire, we have used the technology of protein array analysis to characterize a commercial IVIg preparation by screening its interaction with 9484 proteins of human origin. As a result, we have identified 67 proteins, which are strongly recognized by the IgG present in IVIg. Most of these targets are highlighted for the first time bringing new insight into IVIg mechanisms of action.

Keywords
Immunomodulation; Therapeutic immunoglobulins; IgG self-repertoire; Human proteins
Abbreviations
LIM: named after their initial discovery in the proteins Lin11 Isl-1 Mec-3; PDZ: postsynaptic density protein-95/disks large/zonula occludens-1; PDLIM2: PDZ-LIM domain-containing
Introduction
Intravenous immunoglobulins (IVIg) represent pooled IgG of thousand blood donors; IVIg are replacement immunoglobulins for immunodeficient patients and have positive modulatory effects in autoimmune and inflammatory disorders (reviewed in [1,2]). Their IgG repertoire is polyclonal, representative of both adaptive and natural immunity and contains self-reactive antibodies [3-5]. IVIg contain antibodies directed against several molecules, for example CD4, CD8, HLA, cytokines, T and B cell receptors, heat-shock proteins (reviewed in [6]). Its mechanisms of action have been extensively investigated. IVIg can proceed through direct interaction with immune cells or indirectly by binding to soluble messengers. It can act upon BCR, TCR and Fc receptors activation [1,5] and can also be related to down-modulation of CD16 (FcγRIII) on macrophages in Kawasaki disease [2,7].
 
Until recently, information about IVIg self-reactivity came from studies focusing on particular diseases. Recently, an extensive study based on mass spectrometry analysis have highlighted that IVIg can bind to several intracellular proteins [3]. This group have done proteomic analysis of IVIg interaction on protein extracts prepared from freshly isolated human umbilical vein endothelial cells and from a cell line derived from human laryngeal carcinoma [3]. A total of 96 intracellular proteins, revealed as specific targets for IVIg, were related to various functions such as chaperones, cytoskeletal proteins, DNA, RNA and protein processing, oxydo-reduction, glycolysis as well as growth regulation. Moreover, fourteen of these IVIg-target proteins were underscored in both cell extracts. In line with the current efforts to further characterize IVIg self-reactivity, we have thus performed antibody-profiling based on protein microarray technology.
Materials and Methods
Human protein microarray
Autoreactivity of a commercial intravenous immunoglobulin preparation (IVIg) (GamunexTM, Talecris Biotherapeutics ltd., Toronto, ON, Canada) was determined by Antibody specificity profiling service of Invitrogen (Carlsbad, USA). IVIg were probed by Invitrogen at 0.1 μg/ml and 1.0 μg/ml. A total number of 9484 human proteins were screened using the ProtoArray Human Protein Microrarrays v5.0. Data analysis was done by Invitrogen using ProtoArray Prospector software. Average background fluorescence values were respectively 107 and 112 (RFU; relative fluorescence unit). The three criteria, according to Invitrogen profiling service experimental design, permit to determine the more significant interaction between IVIg and the different proteins. Briefly, the negative control RFU for each protein was determined by incubation with probing buffer in absence of IVIg and detection by the goat anti-human IgG. Firstly, for each of the 9484 proteins, the IVIg signal value had to be greater than 2000 RFU and more than 2-fold higher than the negative signal. Secondly, the second negative control signal value had to be less than 1000 RFU and thirdly the replicate spot coefficient of variation (CV) had to be less than 50%.
Results and Discussion
Among the 9484 proteins tested, IVIg have a significant interaction with 67 peptides from 66 proteins based on the three criteria described above (Table 1). As previously reported by Bussone et al. [3], most of the interactors were of intracellular origin, except for CD16a, whose two independent peptides were recognized by IVIg. Among these interactor-proteins, we also spotted PDZ-LIM domain-containing protein 2 (PDLIM2) and LIM domain protein 1, which was one of the 96 proteins identified by Bussone’s group, but was not included in their final list of 14 targets. Interaction of IVIg with CD16a (FcγRIIIa receptor) is the only one linked to an IVIg-related disease effect. Indeed, down-modulation of CD16a has been linked to IVIg treatment of patients with Kawasaki disease (KD) [8]. However, IVIg self-reactivity has been shown to target many other proteins present in the 9484 protein arrays, namely heat shock proteins, interleukin- 1β, HLA-DR, CD64 and even influenza A virus antigen H3N2. In our opinion, the absence of reactivity for these well-known antigens appeared unlikely. IVIg repertoire have been estimated to recognize up to 107 antigenic epitopes [9], suggesting that less than 1000 antibody molecules might be available for each epitope when using IVIg at 1.0μg/mL. We then reconsidered the protein array data interpretation by using criteria to include as a positive internal control the reactivity against H3N2. We thus determine that he signal used for IVIg must be higher than 50 RFU, the negative signal must be higher than 1 RFU (to exclude negative values) and the ratio between those two signals must be higher than 5. Finally, the CV for duplicate must be less than 15%. Using these criteria we have observed that IVIg (1.0 μg/ml) was able to interact with 776 peptides, which included all 67 proteins listed in Table 1 (Supplemental data Table 1). Based on these criteria, IVIg were indeed found reactive for influenza A antigen (H3N2) [10,11], which was the only non-human antigen present in this array, showing 526 ± 7% RFU, which was 44-fold stronger than the negative signal. Similarly, we obtained acceptable reactivity for HLA-DR (59 ± 10% RFU; 39-fold), thyroglobulin (205 ± 10% RFU; 26-fold), CD64 ((755 ± 10%, 23-fold), interleukin-1β (251 ± 10%, 15-fold), lamin A/C (277 ± 14%, 8-fold) and ferritin (1177 ± 10%, 5-fold), which are all related to immunomodulatory properties of IVIg [2,3,7,8,12-14]. Downmodulation of CD16a and CD64 has been reported by many studies following IVIg therapy of patients suffering from KD and our results suggest that anti-CD16a and anti-CD64 antibodies could be involved in this mechanism. Overall, our observations bring at least 66 new candidates as strong IVIg interactions and also suggest that IVIg selfreactivity is still underestimated when considering the possibility of 776 protein targets underscored when using criteria based on previous observations. Finally, we hope that this information will be helpful for all groups working on IVIg by comparing their observations with our data, which might expose new routes for IVIg mechanisms of action.
Acknowledgements
The authors are grateful to Rayelle Itoua Maïga, B.Sc. and Éric Petitclerc, Ph.D. for critical review and manuscript editing.
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