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. 2013 Feb 19;110(8):2993-8.
doi: 10.1073/pnas.1213737110. Epub 2013 Feb 4.

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Yariv Wine  1 , Daniel R Boutz, Jason J Lavinder, Aleksandr E Miklos, Randall A Hughes, Kam Hon Hoi, Sang Taek Jung, Andrew P Horton, Ellen M Murrin, Andrew D Ellington, Edward M Marcotte, George Georgiou
Affiliations

Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response

Yariv Wine et al. Proc Natl Acad Sci U S A. .

Abstract

We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic of the workflow for serum Ig deconvolution. (Left) V gene repertoire sequencing pipeline: total RNA from desired B-cell subpopulations is reverse transcribed and amplified by 5′ RACE with IgG-specific (VH) or Igκ/Igλ-specific (VL) primers and sequenced by Roche 454 sequencing. Reads are processed bioinformatically to obtain a database of unique V genes and their relative transcript abundances. The V gene database is used to interpret the MS spectra. (Right) F(ab)2 purification and proteomic pipeline: F(ab)2 fragments from IgG are prepared and subjected to antigen-affinity chromatography. Proteins in the eluent, flow-through, and wash buffer are denatured, alkylated, proteolyzed, and resolved by high-resolution LC-MS/MS. Full-length V genes containing the identified iCDR3 peptides are then determined from the repertoire database. (Lower) Antibody production and validation: synthetic VH genes are assembled into an scFv library using the VL cDNA, then antigen-specific antibodies are isolated by two to three rounds of phage panning and characterized for antigen affinity.
Fig. 2.
Fig. 2.
Immunoglobulin heavy chain variable region (VH gene) and circulating antibody repertoire characteristics. (A) Wu-Kabat variability plot representing the variability of the VH genes is shown on a residue-by-residue basis; (B) bar graphs representing VH germ-line family use (Left) and JH germ-line family use (Right) in the VH DNA repertoire determined by 454 sequencing of cDNA from CD138+ bone marrow plasma cells (n = 4729 VH genes, blue bars), peripheral B cells (n = 2788 VH genes, red bars), or from antibody proteins identified by proteomic analysis of the serum affinity purified IgGs (n = 334, green bars) for the CCH rabbit.
Fig. 3.
Fig. 3.
Identified iCDRH3 peptides from affinity chromatography and alignment of corresponding CDRH3s. (A) Histogram showing frequencies of identified informative peptides corresponding to a unique clonotype (V genes with same VH, JH CDRH3 sequence and 80% homology in the CDRH3) in the antigen-affinity chromatography elution, flow-through, and wash fractions. (Inset) Magnified histogram of the top 15 highest count unique peptides detected in the antigen-affinity chromatography elution, flow-through, and wash fractions. Peptide IDs are ranked by relative abundance in elution. Identified peptides in the affinity chromatography elution fraction that are found overwhelmingly in the flow-through and wash buffer fractions likely correspond to antibodies that bind antigen very weakly or nonspecifically. (B) Pairwise alignment of CCH-immunized rabbit CDRH3s in the antigen-specific serum IgG repertoire and observed exclusively in the affinity chromatography elution. The dendrogram shows hierarchical clustering (based on pairwise sequence alignments at the amino acid level) of CDRH3 sequences detected in the elution at >10-fold higher number of counts relative to the affinity chromatography wash and flow-through. (Numbered sequences represent VH synthesized for binding validation.)
Fig. 4.
Fig. 4.
Competitive ELISA of full-length IgG containing the VH genes corresponding to selected abundant iCDRH3s (Table 1) identified in the CCH immunized rabbit. Antibodies identified using the proposed methodology (Fig. 1) were shown to display subnanomolar affinities. The expression yield of the rabbit IgG-6 antibody in HEK-293 cells was too low for accurate quantitative affinity measurements.
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