Antibodies are glycoproteins secreted by specialized B lymphocytes that serve as the first response against infection. They comprise one of the main effectors of the adaptive immune system.

The high degree of affinity and specificity of antibodies towards antigens led to ubiquitous use of antibodies in life science and medical research. Antibodies can be monoclonal or polyclonal.

Monoclonal antibodies are produced by injecting an antigen into a host animal to initiate a humoral immune response. In most procedures, spleen cells from these hosts are fused in vitro with cultured malignant myeloma cells.

Unique cell clones are isolated and those that survive the fusion step are known as hybridomas. Hybridomas are immortal because of their myeloma characteristics and are easily propagated in culture.

Monoclonals are homologous to natural immunoglobulins from the source immunized animal, but unlike polyclonal antibodies purified from serum, they are specific to a single epitope and provide a stable long term supply of produced by hybridomas in vitro.

Invitrogen™ rabbit recombinant antibodies are derived from rabbit monoclonal antibody-producing cell lines by isolating and cloning the specific antibody heavy and light chain DNA sequences. These recombinant cloned antibodies are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance. 

ABfinity™ technology

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

ABfinity antibodies are 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.

The scheme below demonstrates how ABfinity monoclonal rabbit antibodies are produced. 

abfinity 1

Recombinant rabbit monoclonal antibody production.

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 below shows western blotting results achieved using independent lots of an ABfinity antibody, and its graphical representation.

abfinity antibodies fig1

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).

Figure 2 shows the lot-to-lot consistency in immunocytochemistry, and its graphical representation.

immunocytochemistry fig2

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).

The next figure shows the same results in flow cytometry. 

flow cytometry fig3

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).

Sensitivity and Specificity

The ABfinity 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. 

sensitivity specificity fig4

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.

The next Figure demonstrates the same results in immunocytochemistry. Flow  cytometry (results not shown) revealed similar results, indicating the sensitivity and specificity of ABfinity antibodies.

abfinity fig5

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.

Antibody validation

ABfinity antibodies are validated and characterized by multiple applications.

Examples of their application are shown in the following Figures.

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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. Immunohistochemistry of human oesophagus carcinoma tissue labeled with AKT [pS473] - ABfinity Recombinant Rabbit Monoclonal Antibody. Formaldehyde-fixed paraffin-embedded (FFPE) human oesophagus 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 tumour cells.

 Find out more about ABfinity antibodies on the Thermo Fisher website

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