Elsevier

Journal of Proteomics

Volume 114, 30 January 2015, Pages 115-124
Journal of Proteomics

Delineating monoclonal antibody specificity by mass spectrometry

https://doi.org/10.1016/j.jprot.2014.11.004Get rights and content

Highlights

  • Fractionated seminal plasma was used for the production of mAbs against antigens of varying abundance.

  • A variety of immuno-MS protocols were established by using four types of solid support.

  • Immuno-MS methodologies facilitated the characterization of the newly developed mAbs.

  • Different immuno-MS configurations displayed various capabilities for a more efficient mAb characterization per instance.

Abstract

Generation of monoclonal antibody (mAb) libraries against antigens in complex matrices can prove a valuable analytical tool. However, delineating the specificity of newly generated antibodies is the limiting step of the procedure. Here, we propose a strategy for mAb production by injecting mice with complex biological fluid and mAb characterization by coupling immunoaffinity techniques with Mass spectrometry (immuno-MS). Mice were immunized against fractionated seminal plasma and mAbs were produced. Different immuno-MS protocols based on four types of solid support (i.e. polystyrene microtiter plates, NHS-activated agarose beads, tosyl-activated magnetic beads and MSIA™ pipette tips) were established. A well-characterized mouse monoclonal anti-KLK3 (PSA) Ab was used as a model to evaluate each protocol's robustness and reproducibility and to establish a set of criteria which would allow antigen characterization of newly developed Abs. Three of the newly generated Abs were analyzed using our optimized protocols. Analysis revealed that all assay configurations used were capable of antibody characterization. Furthermore, low-abundance antigens (e.g. ribonuclease T2) could be identified as efficiently as the high-abundance ones. Our data suggest that complex biological samples can be used for the production of mAbs, which will facilitate the analysis of their proteome, while the established immuno-MS protocols can offer efficient mAb characterization.

Biological significance

The inoculation of animals with complex biological samples is aiming at the discovery of novel disease biomarkers, present in the biological specimens, as well as the production of rare reagents that will facilitate the ultra-sensitive analysis of the biomolecules' native form. In the present study, we initially propose a general workflow concerning the handling of biological samples, as well as the monoclonal antibody production. Furthermore, we established protocols for the reliable and reproducible identification of antibody specificity using various immuno-affinity purification techniques coupled to mass spectrometry. Our data suggest that processed biological fluids can be used for the production of mAbs targeting proteins of varying abundance, and that various immuno-MS protocols can offer great capabilities for the mAb characterization procedure.

Introduction

Biological samples are deposits of numerous molecules—characteristic to a greater or lesser extent of the matrix and its functions [1]. In other words, these molecules could provide in-depth biological information and/or associate with disease development and progress. Although ever going technological progress, which promises better analytical procedures, polyclonal and monoclonal antibodies remain important tools for the analysis of biomolecules. Research antibodies are produced by inducing immune response to a host animal against an immunogen representing the human protein. It is evident that the utility of the Ab is related to the high resemblance of the immunogen to the native protein.

High throughput generation of large monoclonal antibody (mAb) libraries against diverse antigens in their native form has been proposed by immunization of mice with the complex biological sample the antigens exist [2]. Until recently, the large-scale specificity characterization of monoclonal antibodies presented a major bottleneck, limiting mAb development to single (recombinant) protein or peptide immunizations. This problem was recently tackled by the advent of immuno-precipitation methods coupled with MS-based proteomics (generally known as immuno-MS methods). These methods promise to revolutionize the field by allowing rapid characterization of the antigen specificity of multiple antibodies [3], [4], [5], [6].

The success of an immuno-MS method depends on the choice of the affinity reagent, the proper separation of affinity reagent-target analyte complex from contaminants, and the suitable recovery of the bound analyte for MS identification. To date, several different immuno-MS formats have been reported, depending on the nature of the affinity-based enrichment used. Other than the classical antibody-based formats, several alternative affinity reagents have also been recently proposed, including DNA aptamers, engineered polypeptides and inorganic molecules [7]. Despite these advances, the classical antibody immobilization on a solid support (beads, plates, columns) still represents the most-established approach to affinity purification.

Several parameters can have a direct effect on the affinity of an individual antibody–antigen interaction, such as the nature of the solid support and the underlying chemistry of each interaction (e.g. Protein A/G–Fc, streptavidin–biotin or covalent binding via NH2 or COOH groups, directed or random binding) [8], [9], [10], [11], [12], [13], [14]. Therefore, the selection of the appropriate set-up of an immuno-MS method should reflect the intended objective of each study. Towards Ab-antigen characterization, three main types of immuno-MS configurations have been described so far: i) polystyrene-based (e.g. microtiter plates), ii) bead-based (e.g. sepharose or magnetic-beads) and iii) MSIA™-tip format [5], [15], [16], [17], [18], [19], [20].

This study investigated the efficiency and robustness of these methods in determining the specificity of antibodies generated by immunizing mice with complex biological fluid. It should be noted that these configurations were used to pull down intact antigens.

As the biological matrix under investigation we decided to work with seminal plasma. Seminal plasma is a rather complex biological fluid, comparable to blood, which seems to be a source of promising biomarkers for the male reproductive system disorders [21]. Furthermore, our lab has worked extensively with seminal plasma, in a long-lasting attempt to decipher its proteome [22], [23], [24].

One of the most abundant components (> 100 μg/ml) of seminal plasma is the prostate-specific antigen (PSA or KLK3)—a member of the kallikrein-related peptidases family. KLK3 is secreted by epithelial cells of the prostate gland and is involved in semen liquefaction [25]. We used a commercial anti-KLK3 (PSA) Ab as a model, in order to optimize the performance characteristics of each immuno-MS set-up, and to develop a set of decision rules, applicable to all configurations. We then successfully implemented these rules for the identification of the target antigens of three unknown antibodies, developed by immunizing mice with fractionated seminal plasma (SP). Technical insights on the innate characteristics of each of these immuno-MS methods are provided.

Section snippets

Fractionation of seminal plasma prior to animal immunization

Seminal plasma samples were pooled (N = 10). Samples were obtained after informed consent and institutional review board approval (Mount Sinai Hospital, Toronto, ON, Canada). Prior to animal injection, samples were subjected to a three-step chromatographic fractionation, including: i) initial FPLC fractionation using a HiTrap Q column (GE Healthcare Life Sciences, Baie d'Urfe, QC, Canada), ii) a higher-resolution fractionation step, using a Source 15Q column (GE Healthcare) and iii) HPLC size

Method optimization using a commercial anti-KLK3 monoclonal antibody

Anti-KLK3 Ab and non-specific mouse IgG (negative control) were coupled onto the four different solid surfaces (polystyrene plate, NHS-activated sepharose beads, magnetic beads and MSIA™ pipette tips). Subsequently, the solid phases were incubated with serial dilutions of seminal plasma. Captured proteins were examined by mass spectrometry as described in Methods. In all cases, the same amount (50 fmol) of heavy-labeled KLK3 proteotypic peptide (LSEPAELTDAVK) was added as an internal standard.

Discussion

This study examines the feasibility and robustness of currently available MS-based technologies to determine the antigen specificity of newly generated Abs. As a proof-of-concept, we selected a commercial anti-KLK3 antibody, which was utilized for immuno-MS protocol optimization using: 1) polystyrene plate formats, 2) NHS- and magnetic beads, and 3) MSIA™ pipette tips. These protocols were then used for the characterization of new Abs, developed in mice by injecting a fraction of human seminal

Conflict of interest

The authors declare no competing financial interest.

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    These authors contributed equally to this work.

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