Advantages of RabMAbs

As monoclonals, RabMAbs provide no or low background signals in various applications. As they are more specifically detecting one target epitope, they are less likely to cross-react with other proteins.

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  Western blot analysis on (A) MCF-7, (B) Ramos, (C) SW480, (D) Molt-4, and (E) Jurkat, and (F) HeLa cell lysates using anti-MCM2 RabMAb (ab108935)

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  Immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma using anti-c-Myc RabMAb (ab32072)

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  Immunofluorescent staining of HeLa cells using anti-Phospho-AMPK alpha 1 (pS496) RabMAb (ab92701).

High specificity and novel epitope recognition of rabbit antibodies translates to success with recognition of post-translational modifications such as phosphorylation, methylation, acetylation, sumoylation and other types of modifications. Additionally, many small compounds and peptides do not elicit a good immune response in mice but do so in rabbits. See below for examples of Histone H3 modifications specific RabMAbs.

• Acetylation

  

  Western blot analysis on C6 cell lysate using anti-Histone H3 Acetyl-K56 RabMAb (ab76307). Lane (1) untreated; lane (2) treated with TSA

• Methylation

  

  Western blot analysis using anti-Histone H3 Dimethyl (K4) RabMAb (ab32356). Lane (1) Hela cell lysate; lane (2) recombinant Histone H3 (non-methylated)

• Phosphorylation

  

  Western blot analysis on NIH 3T3 cell lysate using anti-Phospho-Histone H3 (pS28) RabMAb (ab32388). Lane (1) untreated; lane (2) treated with FBS and Calyculin A

RabMAbs offer increased sensitivity with no loss of specificity, making them ideal for demanding applications like IHC on FFPE tissues. RabMAbs permit higher working dilutions (5 - 10X on average) and can be used in various tissue fixations such as FFPE at minimal level of pretreatment. Additionally, used along with a mouse monoclonal, one can perform dual staining with two monoclonal antibodies for high quality double staining on the same tissue sample.

• Her RabMAb

  

  3ng / mL

• Rabbit Polyclonal Antibody (Vendor A)

  

  20ng / mL

• Mouse Monoclonal (Vendor B)

  

  30ng / mL

A comparison of a Her2 RabMAb with leading commercially available Her2 rabbit polyclonal (vendor A) and mouse monoclonal (vendor B) antibodies on FFPE human breast carcinoma tissue. Our recommended IHC protocol and dilution factor were used for each case, the antibody concentrations used are listed below the images for each stain.

Antibody affinity is typically represented by the equilibrium dissociation constant (K_D), a ratio of k_off/k_on, between the antibody and its antigen, where a lower K_D value suggests higher affinity relationship (5). While most therapeutic antibodies have a K_D value in the nanomolar range (K_D =10^-9 M), RabMAbs consistently demonstrate higher affinity, with the K_D values which can often reach the picomolar level (10^-12 M), effectively eliminating the need for further affinity maturation (6).

Antibody affinity comparison by K_D value for various RabMAbs vs. popular therapeutic antibodies

Antibody affinity comparison by K_D value for a typical RabMAb vs. traditional mouse monoclonal antibodies.

RabMAb technology offers a high diversity of B cells and an abundance of splenocytes, which equate to more success in generating antibodies with higher specificity. The example below shows that the Anti-Progesterone RabMAb does not cross-react with closely related analogues of progesterone.

Additionally, a RabMAb’s superior specificity means that you have high quality antibodies which recognize subtle changes in epitopes such as those created upon protein cleavage.

PARP specific RabMAbs

The rabbit’s lower immune dominance and larger B-cell repertoire result in greater epitope recognition. See below for a comparison of rabbit and murine immune responses which shows that rabbit antisera recognize more epitopes than mouse antibodies in a Western Blot analysis.

Additionally, major relevant human protein targets are highly conserved between mouse and human. Therefore, these proteins tend to be less immunogenic when using mouse or rat as a host. Using a rabbit as a host, with its unique mechanism of immune diversification and affinity maturation, RabMAbs can be produced to a wider range of epitopes, allowing discovery of more novel antibody targets.

Each RabMAb produced is tested in multiple applications (WB, IHC, ICC/IF, IP and FACS) and multiple species (Human, Mouse and Rat) before release. For IHC, all RabMAbs are tested on FFPE human tissue array for more accurate verification of antibody sensitivity and localization.

• A

  

• B

  

• C

  

• D

  

Validation data for Caspase-3 (Pro) RabMAb (ab32150) on A) WB on HeLa cell lysate, B) IHC on FFPE cervical carcinoma tissue, C) FACS on Jurkat cells and D) ICC on HeLa cells.

With the rabbit as a host, RabMAbs are ideal candidates for developing high quality antibodies to mouse targets. RabMAb technology can produce monoclonal antibodies to mouse targets which have been less immunogenic in mouse or rat. Additionally, with a RabMAb, one can use a monoclonal antibody in mouse model without the issue of non-specific signal due to native mouse Ig cross-reactivity. See below for highlights of RabMAbs tested on FFPE mouse tissue.

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  Paraffin-embedded mouse intestine using anti-EGFR Phospho (pY1068) RabMAb (ab40815)

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  Paraffin-embedded mouse smooth muscle using anti-Calponin-1 RabMAb (ab46794)

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  Paraffin-embedded mouse colon using anti-Cytokeratin-8 RabMAb (ab53280)