Authors – Karuppuchamy, Thangaraj, Vartak-Sharma, Neha, Dalmeida, Rimple, kannadasan, kaliyamoorthy and Sridharan, Haripriya
Antibodies are some of the most commonly used reagents in basic and preclinical research, and it is crucial to have specific and highly sensitive antibodies in order to correctly interpret experimental results. Customers are increasingly demanding recombinant antibodies as these offer several advantages compared to traditional polyclonal or hybridoma antibodies, such as reproducibility in performance, sustained availability, and animal free production. A unique benefit of recombinant antibodies is that they are amenable to rational engineering strategies to confer desired functional benefits. Recent advances in the field of antibody engineering have allowed for production of highly specific recombinant antibodies with different modifications and formats. While antibody engineering has been exploited extensively for the development of therapeutic reagents with enhanced immuno – protective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against specific cancer or immunology targets. Advanced antibody engineering techniques have extensive applications for research use in the fields of immunology, biotechnology, and molecular biology. Here we will discuss the potential of engineering of recombinant research-use antibodies and illustrate the benefits through selected case studies of antibody engineering that are designed to drive increased sensitivity in immunoassays, resulting in detection of less abundant endogenous targets and improving signal to noise ratios.
One of the most important characteristics of a good antibody is its sensitivity, i.e., the measure of the goodness of fit between the antibody paratope and the corresponding antigenic epitope, (Candler et al., 2006). A highly sensitive antibody has several benefits, such as the ability to detect low abundance targets and can be used in multiplexing immuno assays. Antibody sensitivity can be increased by affinity maturation of the antibody clone, which involves mutations of the antigen binding site or paratope. However, affinity maturation must be tailored for each antibody under consideration and therefore not a scalable solution.
On the other hand, IgG Fc engineering is one attractive, robust strategy to maximize the efficacy and overcome the drawbacks associated with weak monoclonal antibodies. Using our proprietary technology, we have engineered the Fc region of the rabbit recombinant monoclonal antibody (Figure 1) and validated in multiple immunoassays.
Figure 1. Schematic representation of the wild type (WT) and engineered antibody
The performance of engineered antibodies was tested in a western blot application. Across the different cell and/or tissue models tested, both Parkin and OCT4 engineered recombinant rabbit monoclonal antibodies (Product #740019R and 740020R) demonstrated a two-fold sensitivity enhancement over the wildtype (WT) parental antibodies (Figure 2). While the actual fold change may vary between experiments, cell/tissue models, and the antibody concentrations used, the results were significant as determined by the independent student’s T test respectively (**p<0.01, ***p<0.001).
Figure 2: Recombinant antibody testing data in western blot for Parkin and OCT4 Recombinant Rabbit Monoclonal Antibodies (Product #740019R and 740020R, respectively). Western blot analysis was performed on whole cell extracts of SH-SY5Y (A) and NTERA-2 cells (B). The blots were probed with wild type (WT) and engineered Parkin or OCT4 Recombinant Rabbit Monoclonal Antibody [Product #740019R (1 µg/ml) or 740020R (0.5 µg/ml)]. A ~48 kDa band corresponding to Parkin and ~38 kDa band corresponding to OCT4 were observed. (C) Scatter plot analysis of data from multiple western blot experiments showed significant fold enhancement of signal with engineered antibodies compared to wild type (WT). **p<0.01, ***p<0.001
Like western blotting, the performance of engineered antibodies was further tested in immunocytochemistry application (Figure 3). While the WT Parkin antibody failed to detect Parkin, the engineered Parkin antibody detected Parkin in the expected cytoplasmic localization (Figure 3A). Engineered OCT4 antibody demonstrated a ~1.5-fold increase in signal to noise over parental antibody even when used at 1/8th the concentration of the parental WT antibody (Figure 3B).
Figure 3: Recombinant antibody testing data in immunocytochemistry for Parkin (A) and OCT4 (B) Recombinant Rabbit Monoclonal Antibody (Product # 740019R and 740020R). Immunocytochemistry analysis of Parkin and OCT4 was performed using 70% confluent SH-SY5Y and NTERA-2 cells respectively. The cells were probed with wild type (WT) and engineered Parkin or OCT4 Recombinant Rabbit Monoclonal Antibody at the indicated concentrations (Product # 740019R or 740020R). For Parkin, cells were also stained for nuclei, using SYTOX™ Deep Red Nucleic Acid Stain (Product # S11381).
Furthermore, along with the increased sensitivity, there was no compromise on the specificity of the engineered antibodies. siRNA-mediated knockdown of Parkin and OCT4 signal in western blot and immunocytochemistry demonstrated the specificity of the engineered Parkin and OCT4 antibodies, respectively. (Figure 4 and 5).
Figure 4: Specificity testing data in western blot for Parkin and OCT4 Recombinant Rabbit Monoclonal Antibody (Product #740019R and 740020R). Antibody specificity was demonstrated by siRNA-mediated knockdown of the target protein. (A) SH-SY5Y cells were transfected with Parkin siRNA and a decrease in signal intensity was observed in western blot application using engineered Parkin Recombinant Rabbit Monoclonal Antibody (Product #740019R). (B) NTERA-2 cells were transfected with OCT4 siRNA and a decrease in signal intensity was observed in western blot application using engineered OCT4 Recombinant Rabbit Monoclonal Antibody (Product #740020R).
Figure 5: Specificity testing data in immunocytochemistry for OCT4 Recombinant Rabbit Monoclonal Antibody (Product #740020R). Antibody specificity was demonstrated by siRNA-mediated knockdown of the target protein. NTERA-2 cells were transfected with OCT4 specific siRNA and a decrease in signal intensity was observed in immunocytochemistry application using engineered OCT4 Rabbit Recombinant Monoclonal Antibody (Product #740020R).
To demonstrate the wide applicability of the engineered antibodies, different secondary antibodies such as superclonal HRP, polyclonal HRP and poly HRP antibodies were used in western blot application. The fold enhancement observed with engineering was retained across the different secondaries tested. This demonstrates the ease with which our engineered recombinant antibodies can be incorporated into existing workflows with minimal to no changes to other reagents being used.
Figure 6. Bar graph analysis of western blot data obtained from Parkin (A) and OCT4 (B) Recombinant Rabbit Monoclonal Antibody (Product #740019R and 740020R). Engineered antibodies showed fold enhancement across different secondaries compared to wild type (WT) antibody.
Utilizing our proprietary engineering technology, we have further expanded the application coverage from just being functional in western blot application to now being functional in immunocytochemistry as well as immunohistochemistry (IHC) and flow cytometry (Figures 7, 8 and 9).
Figure 7. Immunohistochemical analysis of Parkin was performed using formalin-fixed paraffin-embedded mouse kidney (A), mouse spleen (B), human cerebral organoids (C) and rat brain (D) tissue sections. The sections were probed with engineered Parkin Recombinant Rabbit Monoclonal Antibody (Product #740019R) or corresponding wild type (WT) Parkin antibody.
Figure 8. Flowcytometry analysis of Parkin was performed in PC-12 cells. Cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Product #88-8824-00) and then stained intracellularly with 2 µg of Parkin wild type (WT) or Engineered Parkin Recombinant Rabbit Monoclonal Antibody (Product #740019R) antibody. Unstained samples were used as a control. ***p<0.001
Figure 9. Flowcytometry analysis of OCT4 was performed in NTERA-2 cells. Cells were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (Product #00-5523-00) and then stained intracellularly with 0.25 µg of OCT4 wild type (WT) or Engineered OCT4 Rabbit Recombinant Monoclonal Antibody (Product #740020R). Unstained samples were used as a control. *p<0.05
Our proprietary engineering in rabbit recombinant monoclonal antibodies have demonstrated exceptional sensitivity and excellent signal to noise across different immunoassays such as western blotting (WB), immunocytochemistry/immunofluorescence (ICC/IF), immunohistochemistry (IHC), and flowcytometry. Lowering the limits of detection by increased sensitivity of these engineered antibodies enables the study of low abundant targets such as Parkin and OCT4 in immunoassays, helping save precious samples enabling a cost advantage to the customer. Engineering of Fc domain of IgG without altering the antigen binding domain (Fab) and function, thereby allowing for universal and scalable methods, which can be applied across several antibodies. The Engineered antibodies are for Research Use Only (RUO). Not for use in diagnostic procedures.
Learn more about Invitrogen Recombinant antibodies at thermofisher.com/recombinantantibodies.
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