Letter pubs.acs.org/jpr
Chicken Immune Responses to Variations in Human Plasma Protein Ratios: A Rationale for Polyclonal IgY Ultraimmunodepletion Sock Hwee Tan, Amit Kapur,† and Mark S. Baker* Department of Chemistry and Biomolecular Sciences and Biomolecular Frontiers Research Centre, Macquarie University, NSW, 2109, Australia ABSTRACT: Chicken IgY responses against mixtures of six high abundance human plasma proteins were studied across a range of abundances from 1:1 for all six proteins to where one protein predominated above the other five by ≤1000 fold. The ability of any protein in that mixture to mount an IgY response varied. In the 1:1 mixture, human IgG produced the highest and α1antitrypsin the lowest individual responses. However, increasing relative abundance of any protein over others increased the total IgY response (i.e., sum of all responses to each antigen) above those obtained for 1:1 ratios. Increasing relative abundance of any protein over others in the mixture (e.g., 1:10, 1:100 and 1:1000) resulted in variations in response to both the overexpressed antigen and to the other five “constant” proteins. The overriding trends were that as individual proteins became relatively more abundant, they elicited higher IgY responses, while the remaining proteins elicited constant or even decreased responses. This study demonstrates that the ability to mount an IgY response to complex plasma protein antigens (e.g., human plasma) is conclusively driven by a combination of individual antigen immunogenicity and the relative abundance of components within any mixture.
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TO THE EDITOR, Human plasma has been proposed to contain a rich source of biomarkers for early disease diagnosis, prognosis and patient response to therapy. However, plasma protein biomarker discovery is routinely hindered by the presence of many highly abundant proteins, such as albumin and IgG, which occupy most of the proteomics discovery space - the so-called challenging dynamic range issue where the range of protein concentration can be greater than 12 orders of magnitude.1 To circumvent this problem, multidimensional separation strategies coupled with tandem mass spectrometry (LC−MS/MS) have been used to enhance detection of lower abundance proteins and improve plasma proteome coverage. Commonly used methods include the immunodepletion of highly abundant plasma proteins and fractionation of the remaining proteome (at protein/peptide level) into smaller subsets prior to LC− MS/MS.2−4 At present, several immunodepletion products (e.g., MARS-6, MARS-7, MARS-14, Seppro IgY14 and ProteoPrep 20) are commercially available to capture up to 20 high abundant human plasma proteins in a single step. These products have been widely employed to reduce the dynamic range of plasma protein concentrations and increase coverage.3,5−7 However, increasing the number of depleted proteins from 12 to 20 has been found to have minimal impact on “simplifying” the plasma proteome.8 This implies that both the bleed-through of high abundance (HAP) and presence of numerous moderately abundant proteins (MAP) continue to dominate the remaining immunodepleted proteome, masking detection of potential low abundance (LAP) biomarkers. The introduction of the novel immunoaffinity column Seppro IgY based product SuperMix provided new insights © 2012 American Chemical Society
into achieving greater resolution of the human plasma proteome. This tandem immunoaffinity depletion (IgY12/ SuperMix) system was shown to be capable of removing 12 of the most abundant and approximately 50MAPs from plasma.9,10 This advance led to a substantial improvement in protein coverage, whereby it was then possible to detect proteins found that at ang/L level.10 These findings collectively suggested the possibility that polyclonal IgY immunoaffinity columns could potentially remove hundreds of high and medium abundance proteins (i.e., both HAPs and MAPs). The use of the chicken host to produce polyclonal antibodies (IgY) against complex mammalian antigens offers several advantages but has not been exhaustively studied to date. First, the deep phylogenetic distance between chicken and humans means that immunogenicity is far more potent than in for example other mammals like goat, horse, rat or rabbit11 Second, the subsequent purification of IgYs from the egg yolk has recently been shown to be considerably easier, as well as much more noninvasive than using mammals.12,13 Finally, the amount and specificity of IgY antibodies produced from chickens is greater than that of mammalian hosts. Raising polyclonal chicken IgYs against mixtures of human proteins has previously been reported to effectively immunodeplete many highly abundant proteins from plasma.9,14 However, the factors that might influence the likelihood of any plasma protein antigen mixture in inducing effective chicken immune responses has not yet been studied, nor has the effect of relative abundance in that mixture been tested. We Received: August 2, 2012 Published: October 18, 2012 6291
dx.doi.org/10.1021/pr300717b | J. Proteome Res. 2012, 11, 6291−6294
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Figure 1. Total antibody response (TAR) detected in chickens (two per group) postimmunized with different ratios of protein mixtures.
in TAR when the ratio of any protein increased from 100-fold excess to a 1000-fold excess over the other plasma proteins. A possible explanation for this observation could be that antibody response is then dominated by a single protein present in 1000fold excess. In this case, all antibody responses resulting from other less abundant proteins were found to be near trivial and therefore lead to decreased overall TAR. This data suggests that the relative concentration ratios in the mixture play a vital role in determining the antibody response elicited by any complex antigen. As the data shows, the TAR for each group differed. Hence, specific IgY activity was then expressed in term of relative antibody response (RAR), which refers to the ratio of antibody response against a specific antigen relative to the total antibody response for the complex protein mixture. When the 6 proteins were mixed in an equal ratio (Figure.2), α1antitrypsin elicited
hypothesized that given the great evolutionary distance between humans and chickens, that in mixtures of plasma protein immunogens relative antibody responses might largely be driven by a combination of (i) the relative abundance of any protein in that mixture and (ii) the relative sequence homology of those proteins compared to the hosts counterparts.9 These assumptions are comprehensively tested here. The aim of this study therefore was to investigate the effect of relative abundance of individual combinations of human plasma proteins in complex mixtures to measure their relative effectiveness in inducing chicken IgY responses. These studies were performed primarily in an effort to improve our understanding of the role of protein abundance on the development of the chicken host immune response against human antigens primarily for downstream applications of IgYbased polyclonal technologies (e.g., ultraimmunodepletion). The experimental setup in this study involved mimicking protein abundance in human plasma by mixing six purified human plasma protein antigens (albumin, IgG, α1-antitrypsin, haptoglobin, transferrin and IgA; all at 95% purity and obtained from Sigma, St Louis, MO). These proteins were mixed across a decreasing weight ratio from 1:1 (for all six proteins) to where a single protein predominated above the other five by ≤1000 fold. In detail, 55 weeks old ISA Brown chickens were immunized intramuscularly with antigen preparations (final amount 625 μg), mixed with Freund’s Incomplete Adjuvant over four immunizations. Eggs were collected after the fourth immunization and IgY purified according to our recently described novel method.15 IgY antibody responses were monitored using an indirect enzyme-linked immunosorbent assay (ELISA) format. Briefly, albumin, IgG, α1antitrypsin, haptoglobin, transferrin and IgA were coated onto a 96-well plate as antigens and elicited IgY antibody response generated against each protein was measured by ratio of ELISA absorbance of sample relative to preimmune IgY at 450 nm. In order to determine total antibody response (TAR), antibody responses generated against individual proteins were summed. For example, when the antibody responses for albumin, α1-antitrypsin, transferrin, haptoglobin, IgG and IgA at week 5 were 4.066, 2.729, 2.821, 3.036, 6.558, and 3.664 respectively, the TAR was expressed as the sum of these 6 individual responses (i.e., 22.8). In our study, although the same amount of protein (625 μg) was immunized over 4 distinct immunizations total antibody response (TAR) varied greatly, ranging from 10.8 to 93.3 (Figure 1). A common phenomenon observed was a decrease
Figure 2. Relative antibody response (RAR) of individual proteins detected in chickens (two per group) postimmunized with mixture containing proteins in equal ratio.
by far the lowest relative antibody response, while IgG elicited the highest relative antibody response. This suggested that although proteins used in our study were all of human origin with variable sequence homology to chicken counterpart proteins, significantly different levels of chicken IgY antibody responses were observed between individual immunogens. The highest and lowest sequence homology (Blastp version 2.2.27, Swiss-Prot database) observed between human proteins and chicken orthologs was 51% (serotransferrin, P02787 and ovotransferrin, P02789) and 28% (α1antitrypsin, P01009 and ovalbumin, P01012). Given the apparent lack of correlation between the ability to elicit a strong immune response and immunogen sequence homology to nearest chicken homologue, our data suggests that evolutionary distance between antigen and host’s homologue is not the sole driver of IgY antibody response. In this case, IgG was far more antigenic when presented as an antigen in any complex protein mixture 6292
dx.doi.org/10.1021/pr300717b | J. Proteome Res. 2012, 11, 6291−6294
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Letter
Figure 3. Relative antibody response (RAR) of individual proteins detected in chickens (two per group) postimmunized with different protein mixtures. (A) α1-Antitrypsin was in excess; (B) albumin was in excess; (C) transferrin was in excess; (D) haptoglobin was in excess; (E) IgG was in excess; (F) IgA was in excess.
than any of the other 5 antigens employed. α1-Antitrypsin is a weak immunogen when immunized as a single protein, achieving an antibody response of only 1.97 when a total of 625 μg of protein was administrated (data not shown). However, when α1-antitrypsin was immunized together with other proteins (i.e., albumin, transferrin, haptoglobin, IgG and IgA), a higher antibody response of 7.69 was achieved (data not shown). This result suggested possible improvement of antibody response of poorly immunogenic proteins when coimmunized with other proteins. Out of the 6 proteins, 4 (i.e., albumin, transferrin, haptoglobin and IgA) demonstrated a stepwise incremental increase in RAR as the ratio of them in any mixture increased (Figure 3B−D, F). A small but significant drop in response was observed when the ratio of IgG in the complex mixture was increased to 10-fold at 100-fold excess. However, the RAR rose significantly when the IgG ratio in the mixture was increased to 1000-fold (Figure 3E). Collectively, this data confirms the contention that relative abundance of a protein in a complex mixture is one of the major factors responsible for generating an antibody response
against any proteinin the mixture. In most cases, there was a reasonable correlation between declining RARs with decreasing ratio in the mixture, especially when the relative concentration dropped by ∼3 orders of magnitude. This is a particularly important observation in our understanding of how products like the Seppro SuperMix Columns work. Human plasma proteins, such as interleukins and chemokines, found at concentrations dramatically lower (i.e., >3−4 logs) than highly abundant proteins present in a complex mixture are unlikely to generate any significant antibody response. Therefore, the possibility of inadvertently depleting low abundance proteins (i.e., cytokines and biomarkers) should be avoided. In conclusion, this study further supports the exploration of suites of antiplasma polyclonal chicken IgY antibodies against complex human protein antigens and subsequent use of systems derived on these IgY preparations for ultradepletion biomarker studies. 6293
dx.doi.org/10.1021/pr300717b | J. Proteome Res. 2012, 11, 6291−6294
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Letter
(14) Rajic, A.; Stehmann, C.; Autelitano, D. J.; Vrkic, A. K.; Hosking, C. G.; Rice, G. E.; Ilag, L. L. Protein depletion using IgY from chickens immunised with human protein cocktails. Prep. Biochem. Biotechnol. 2009, 39 (3), 221−47. (15) Tan, S. H.; Mohamedali, A.; Kapur, A.; Lukjanenko, L.; Baker, M. S., A novel, cost-effective and efficient chicken egg IgY purification procedure. J. Immunol. Methods 2012.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address †
Wollongong Hospital South Eastern Sydney and Illawarra Area Health Service Wollongong, 2500 NSW, Australia Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the ARC Linkage Program (LPO455692) and China-NSW Government Collaborative Research Program for grant funding. S.H.T. thanks Macquarie University for support through a Research Excellence MQRES PhD Scholarship.
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