May 1, 2017 (Vol. 37, No. 9)

John M. Rosenfeld Ph.D. Strategic Marketing Manager Active Motif

AbFlex Recombinant Antibodies and the Sortag-IT Labeling System

What makes a “good” antibody these days? The research community has different criteria based on their uses. Using antibodies for therapeutics is clearly the most demanding application. Therapeutic antibodies are characterized endlessly for their utility. The demands on them are appropriate, as the performance of these reagents have clinical impact and safety hurdles to achieve.

Over the last decade, numerous studies have been published investigating quality of the commercial research reagent antibodies, and in general, customers are frequently dissatisfied with the average commercial product. Most recently, two Nature commentaries that included over 100 signatories communicated a need not only for more specific antibodies, with less variability in performance across lots, but went so far as to demand complete transparency in the process of antibody generation and production (see Bradbury and Plückthun, Nature, Feb. 2015).

This request for recombinant solutions was nothing new to those in the field of antibody generation and manufacturing. These scientists were already driven in this direction by a common need for quality of supply and sustainability of product. At Active Motif, a decision was made to take recombinant antibody production a step further. To not only clone and express the valuable sequences associated with our antibodies, but to also modify the antibody structure to include a unique design of functional sequences useful for a variety of antibody applications. A sharper tool than your average monoclonal antibody in fact. We call these antibodies AbFlex™ recombinant antibodies.

AbFlex—A Flexible, Multi-functional Antibody Tool

AbFlex antibodies are expressed with three distinct molecular tags on the carboxyl terminus of the antibody heavy chain (see Figure 1A). The antibodies are expressed from a murine IgG2a constant heavy and light chain framework, with cloned sequences for antigenic determination fused to this framework. The molecular tags include a sortase A recognition site (LPETG) in the amino terminal “first” position. Sortase A5 is a penta mutant transpeptidase (protein ligase) derived from Staphylococcus aureus. Sortase A5 exceeds the activity of the wild-type sortase A by up to 100-fold.

Combined with the Sortag-IT™ labeling system (see Figure 1B), sortase A5 can install any number of chemical or biological probes via poly-glycine conjugates (see  Figure 1C). Sortase activity enables coupling of these conjugates stoichiometrically and in a site-directed fashion. Operationally, this means there is virtually no chance of over-labeling. Customers can expect to get up to two conjugations at most per antibody molecule specifically at the location of the molecular tags. Given the directed nature of the labeling there is no need to validate the antibody post labeling with respect to concerns for labeling affecting the antigen recognition site, and when using Sortase A5 for labeling, the subsequent tags are cleaved from the molecule.

An avidin tag is also installed in the “middle” position, to facilitate site-directed enzymatic addition of biotin species via BirA activity. Benefits of this enzymatic addition of biotin include controlled installation of a maximum of two biotin molecules per antibody. This eliminates any fear of over-labeling or impacting the antigen recognition sequences and enables more control over site directed orientation of the antibody on solid phases, such as streptavidin-coated plates or beads. The third tag at the carboxyl terminus of the heavy chain is a 6X-Histidine tag, to enable purification by nickel chromatography, or antibody capture via an anti-His tag antibody.

Simply combine your AbFlex antibody, glycine conjugate and sortase A5 for simple labeling. Stop solution and purification recommendations are also provided. Two labels are sufficient for most applications tested, although low-abundance proteins can be detected more easily if a labelled secondary is utilized, allowing signal amplification where necessary.

Figure 1. (A) Schematic of the AbFlex Recombinant Antibody design. (B) Reaction scheme for antibody conjugation with the Sortase A5 enzyme. (C) Labels available for use with the Sortag-IT labeling system.

Consistency, Reproducibility, and Service for Production and Labeling

Evidence from cloning and sequencing a variety of hybridomas reveals multiple transcripts from the fused cell genome, which may result from mixed species in the hybridoma population. In the process of cloning and screening, the antibody in some ways becomes refined in this cloning and selection process, with a higher purity of both heavy and light chains that are co-expressed and purified.

All conversion products are screened in appropriate applications to ensure the quality of the product is as good as the parent antibody from which it was derived. In many cases, the affinity and specificity improves upon conversion, simply because the cloned antibody is truly clonal. Sortase A5 can utilize any conjugate or probe that can be coupled to a series of three or more glycines. Nucleic acid and small molecule conjugation is possible as well.

The AbFlex antibodies have been used in bead and plate-based ELISAs, immunofluorescence assays, and ChIP-sequencing using various conjugation strategies. And given the features available on these antibodies for easy conjugation, secondary antibodies are often no longer needed. Given these are direct conjugates on primary antibodies, the user can also explore more colors in fluorescence labeling experiments, since anti-mouse and anti-rabbit or goat secondary are not usually required.

Once converted, risks of lot-to-lot variability are mitigated. Batches are highly reproducible, and a DNA record of the antibody is stored in the Active Motif archive, available for years to come. Expressed from mammalian cells, these full-length antibodies perform exceptionally and are available for loading conjugates as desired with the Sortag-IT system. Antibody content currently focuses on transcription factors, histone and nucleic acid modifications, and epigenetic effectors.

AbFlex custom service is also available, in cases where a customer might have interest in using their own monoclonal antibody or hybridoma converted into the AbFlex framework, and custom conjugation services are also offered. With AbFlex custom service, a researcher can leverage our scientists to find an antibody of any class that works in their application. Visit us today at www.
active? to learn more.

John M. Rosenfeld, Ph.D. (, is strategic marketing manager at Active Motif.