recombinant antibodies

ABfinity™ Recombinant Monoclonal Antibody Technology

High-quality recombinant antibodies for today and beyond

  • Specific—undergo rigorous validation 
  • High performance—proven consistency from lot to lot
  • Efficient—detect low-level targets, with less sample

View all Recombinant Antibodies

What is ABfinity™ Technology?

ABfinity™ recombinant antibodies, produced by proprietary Life Technologies™ technology, are highly specific and high-quality monoclonal antibodies, unmatched for producing consistent results.

ABfinity™ antibodies are recombinant antibodies developed by immunizing animals, screening for functionality, and cloning the immunogen-specific antibody genes into high-level expression vectors. The antibodies are produced on a large scale by expressing them in mammalian cells, and purifying them with protein A. These recombinant antibodies are expressed in mammalian expression systems, but appear just like their counterparts isolated from serum or produced by hybridomas. Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.

Lot-to-lot Consistency

ABfinity™ antibodies are manufactured by transfecting mammalian cells with heavy and light chain antibody cDNA. This highly reproducible process results in unparalleled lot-to-lot consistency. This consistency saves time and money because experimental conditions do not require revalidation. Figure 1 shows the consistent western blotting results achieved using independent lots of an ABfinity™ antibody, and its graphical representation. Figure 2 shows the lot-to-lot consistency in Immunocytochemistry, and its graphical representation.  Figure 3 shows the same results in flow cytometry.

 

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Figure 1—Lot-to-lot consistency using Western Blot.  HeLa cell extracts separated on reducing gels were probed with four different lots of SMAD2 ABfinity™ Antibody. The concentrations in lanes A, B, and C were 2 μg/mL, 1 μg/mL, and 0.5 μg/ mL, respectively. Primary antibody was detected using WesternBreeze® Chemiluminescent Kit–Anti-Rabbit. Left is quantitative representation of four lots at different concentrations, showing best lot-to-lot consistency (n=4, Error Bars= Standard Error).

 

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Figure 2—Lot-to-lot consistency using Immunocytochemistry. Tissue cultured HeLa cells stained with four different lots of SMAD2 ABfinity™ Antibody.  A through D represents Lots 1 through 4 at concentration of 3 µg/mL, E through H represents Lots 1 through 4, at concentration of 0.5 μg/mL.  Alexa Fluor® 488 goat anti-rabbit IgG at 1:1000 was used as secondary antibody. Left is quantitative representation, of four lots at different concentrations, showing best lot-to-lot consistency (n=4, Error Bars= Standard Error).


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Figure 3
Lot-to-lot consistency using Flow Cytometry. Quantitative representation of the flow cytometry, with four independent lots of SMAD2 ABfinity™ Antibody at various concentrations, showing high lot-to-lot consistency.  HeLa cells were fixed and permeabilized using FIX & PERM® Reagents. SMAD2 ABfinity™ Antibody incubation was followed by Alexa Fluor® 488 goat anti-rabbit IgG (n=4, Error Bars= Standard Error).

Reliable Sensitivity and Specificity

The ABfinity™ platform allows production of antibodies that are more sensitive and specific than those achieved with any other antibody development platform. These antibodies only react with the target of choice, eliminating detection of the wrong signal due to unspecific binding. More highly sensitive antibodies can detect very low-level targets that may be difficult to detect with other antibodies. Plus, precious samples are saved, needing less antibody for detection.

Figure 4 shows a direct comparison of an ABfinity™ STAT4 antibody with the best commercial STAT4 antibodies in western blotting. This comparison includes antibodies from polyclonal, traditional hybridoma monoclonal, and rabbit hybridoma monoclonal platforms.   Figure 5 demonstrates the same results in immunocytochemistry. Flow  cytometry (results not shown) revealed similar results, indicating the sensitivity and specificity of ABfinity™ antibodies.

Western Blotting (Click image to enlarge) 
Figure 4—Superior Western Blotting results obtained using ABfinity™ antibody. The STAT4 ABfinity™ and polyclonal antibodies were used at 2, 1, and 0.5 μg/mL. STAT4 antibodies from other vendors were used at their recommended concentrations. Primary antibody was detected using WesternBreeze® Chemiluminescent Kit–anti-Rabbit.

 

 
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 Figure 5—Enhanced Immunocytochemistry results obtained using ABfinity™ antibody. Immunocytochemistry results while using STAT4 ABfinity™ antibody at 2.5 μg/mL compared with the same antibodies as in Figure 4. STAT4 antibodies from other vendors were used at their recommended concentrations. Alexa Fluor® 488 goat anti-rabbit IgG at 1:1000 was used as secondary antibody.

Extensive Validation and Characterization

ABfinity™ antibodies are validated and characterized by multiple applications. This extensive validation process allows confidence in target specificity, without any need for optimization. Examples of their application are shown in Figures 6
through 9.


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Figure 6—
Flow cytometry of Jurkat cells labeled with AKT [pS473] - ABfinity™ Recombinant Rabbit Monoclonal Antibody. Jurkat cells were incubated with 50 µM PI3K/AKT signaling pathway inhibitor LY294002 (red trace) or without (green trace) for 1 h prior to being fixed and permeabilized using FIX & PERM® reagents. Cells were then stained with 1 μg/test of ABfinity™ Recombinant Rabbit Monoclonal Antibody.  followed by Alexa Fluor® 488 goat anti-rabbit IgG. The blue trace represents secondary antibody alone.

      


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Figure 7—Immunocytochemistry of mouse fibroblasts cells labeled with AKT [pS473] - ABfinity™ Recombinant Rabbit Monoclonal Antibody. Mouse fibroblast cells were treated with (A) or without (B) 10 µg/mL insulin and labeled with rabbit anti-AKT [pS473] (5 µg/mL). Signal is knocked down after incubation with the phosphopeptide used as antibody immunogen (C) but not with the non-phosphopeptide (D). Alexa Fluor® 488 goat anti-rabbit IgG at 1:1000 was used as secondary antibody, nuclei are stained with Hoechst (blue).

 

  


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Figure 8—Immunohistochemistry of human esophagus carcinoma tissue labeled with AKT [pS473] - ABfinity™ Recombinant Rabbit Monoclonal Antibody. Formaldehyde-fixed paraffin-embedded (FFPE) human esophagus carcinoma tissue was labeled with rabbit anti-AKT [pS473] (0.5 µg/mL). Tissues were pretreated with EDTA and detected with SuperPicTure™ Polymer DAB. Images were taken at 20X magnification. Note nuclear and cytoplasmic staining in tumor cells.

 


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Figure 9—Western blot of 3T3 cell lysates labeled with AKT [pS473] - ABfinity™ Recombinant Rabbit Monoclonal Antibody
.  Rabbit anti-AKT [pS473] (0.1 µg/mL) was used to label AKT [pS473] in untreated 3T3 lysates (lane 1) or PDGF treated 3T3 lysates (lane 2).