A control antibody is essential for validating specificity of a testing antibody. A properly chosen control antibody helps to eliminate most if not all difficulties in sorting out specific vs non-specific activities of a testing antibody. Depending upon the nature of antibody studies, considerations other than isotype-match are necessary in order to clearly demonstrate the specificity of the testing antibody. These include off-target antigen reactivity, Fc glycosylation patterns and effector functions, protein intactness, homogeneity in solution, formulation, and endotoxin contents. This blog discusses each one of these considerations to help selecting a proper control antibody for various applications which are summarized in the table above.
1. Off target antigen reactivity
One common practice in choosing a control antibody is to identify one that does not recognize the antigen of the testing antibody (target antigen). It should be mentioned that while such a control antibody does not recognize the target antigen, it may still be reactive to another molecule(s) that is (are) present in the testing material (e.g., on the cell surface or in the cell lysates) and result in significant background in various assays ranging from immunoprecipitation, FACS, to cell-based assays and animal studies. Ideally, a control antibody should not react to an antigen, yet retain all other biochemical and biological characteristics identical, or, minimally, similar the testing antibody. Indeed, AB Biosciences has engineered such antibodies whose CDR regions have been recombinantly silenced and their functional Fc regions for effector functions are fully preserved. Collectively, we named these antibodies Z-MAB® for zero-binding monoclonal antibody. For more information, please go to: https://abbiosciences.com/collections/z-mab.
2. Species of origin and immunoglobulin isotype
Species matches are critical as the commonly used secondary antibody for western blot, ELISA, IP, IHC and FACS are species-specific. In addition, since mouse IgG Fc cross-talks to human Fc receptors and vice versa species matched controls are essential for cell-based assays and animal studies. Similarly, isotype matches are important for the in vitro assays since the overall antigenic epitopes recognized by the secondary antibody can be quite different from isotype to isotypes. In addition, Isotype match is an important factor for consideration in the cell-based assay and in animal studies since each isotypes exhibit an unique binding behavior toward Fc receptors which can lead to very different effector function outputs. In short, a species/isotype matched control antibody can drastically improve our ability to discern the specific vs non-specific activities of a testing antibody.
Depending upon the material sources, the purity, the storage conditions, an antibody preparation may be subjected to proteolytic digestion and resulted in partial or complete loss of the antibody’s functionalities. These include loss of antigen-binding activity, compromised binding by one or more the Fc receptors, inability to activate complements, and reduced half-life in circulation. In addition, even limited proteolytic digestion can altered/reduced antigenic epitopes recognized by the secondary antibody in immunoassays. As such antibody intactness is important in all the assays listed in the above table. All Z-MAB® control antibodies produced by AB Biosciences were quality controlled by analyzing with both reducing and non-reducing SDS-PAGE for verifying the integrity of heavy and light chains as well as the inter-chain disulfide linkages.
4. Fc glycosylation and effector functions
The glycan moiety associated with amino acid Asn 297 in the heavy chain CH2 region defines antibody’s affinity toward Fc receptors, the ability to activate complements, but not its interaction with neonatal receptor (FcRn). As such, loss of this glycan complex by eliminating the NXT/S consensus glycosylation site compromises antibodies’ effector functions including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) without affecting their serum half-life. It is conceivable that control antibody with Fc glycan matched to the testing antibody is absolutely critical for the cell-based assays and for animal studies, but not so for most other in vitro assays such as western blot and ELISA. Also worthwhile to mention is that IgG antibodies missing the fucosyl sugar from the glycan complex associated with Asn 297 exhibit drastically enhanced binding to human FcgRIIIa and mousse FcgRIV. Therefore if the testing antibody is afucosyl, a matched afucosyl control antibody is required for both for cell-based assays and animal studies.
5. Homogeneity by SEC
Antibody preparations often contain aggregated material resulting from insufficient refolding of the low-pH eluent(s) off the affinity column. Since antibody aggregates can potentially present multiple functional variable regions as well as multiple functional Fc domains which result in enhanced antibody bindings (avidity interaction). As such protein homogeneity with single antibody molecular species is the key quality control for testing antibodies. To avoid the complications associated with avidity binding(s) of the control antibody to Fc interacting proteins such as various Fc receptors and complements, the same homogeneity qualification is needed for control antibody preparations. This is particularly critical for IHC staining, FACS sorting, cell-based assays and animal studies. Z-MAB® control antibodies produced by AB Biosciences was sized by SEC chromatography selected for single molecular species in solution (https://abbiosciences.com/collections/z-mab).
Most of the reagent antibodies are prepared in isotonic buffers with neutral pH, commonly the phosphate buffer saline with pH of 7.2-7.4. In this case, the control antibody ought to be prepared in the same buffer system by buffer exchange through dialysis or preferably size-exclusion chromatography. In contrast, for antibody drug candidates, formulation development is required to establish the best buffer systems suited for the unique biochemical properties of these antibodies. Therefore, formulation difference is not important for western blotting, ELISA, IP, IHC, and FACS as the testing antibodies are likely to be extensively diluted. However, formulation is likely to be an important factor for cell-based assay and definitely should be account for in animal studies.
7. Endotoxin content
Endotoxin is the lipopolysaccharides decorated on the outer membrane of the Gram-negative bacteria and is best known for its inflammation inducing activity affecting both cell growth and functions. As such, endotoxin exposure can be a source of significant variability in the results of antibody studies. Control antibody with low endotoxin content is the best way to circumvent the background signals that routinely complicate results from the cell-based assays and animal studies. Please see also “Regarding Endotoxin” at https://abbiosciences.com/blogs/news/regarding-endotoxin.