Rabbit Monoclonal Antibodies

Wong SC, Chan JK, Lo ES, et al. The contribution of bifunctional SkipDewax pretreatment solution, rabbit monoclonal antibodies, and polymer detection systems in immunohistochemistry. Arch. Pathol. Lab. Med. 2007 131 1047-1055. 

Use specific hybridoma to produce Rabbit Monoclonal Antibody in bio reactor... 

Fig. 1.5 The general outline that Epitomics uses for producing a rabbit monoclonal antibody...

The basic principle for making the rabbit monoclonal antibody is the same as for mouse monoclonals. Rabbit fusion partner cells can fuse to rabbit B-cells to create the rabbit hybridoma cells. Hybridomas are then screened to select for clones with... 

This juxtaposes the bcl-1 locus to the immunoglobulin (Ig) gene sequences and leads to deregulation of cyclin Dl. A similar translocation is present in a subset of multiple myeloma but is not present in hairy cell leukemia. The mechanism of overexpression of cyclin-D 1 in hairy cell leukemia is unknown. Detection of the expression of Bcl-1 is less helpful in establishing a diagnosis of mantle cell lymphoma. Several clones are available for the detection of this antigen in formalin fixed paraffin embedded material however, rabbit monoclonal antibodies are the most robust. The level of cyclin-D 1 in mantle cell lymphomas, multiple myeloma, and hairy cell leukemia is much less than in epithelial tumors and even normal tissue and one must use cases of mantle cell lymphoma to successfully establish validation of your selected immunohistochemical platform. 

Powell WC, Hicks DG, Prescott N, et al. A new rabbit monoclonal antibody (4B5) for the immunohistochemical (IHC) determination of the HER2 stams in breast cancer Comparison with CBll, fluorescence in sim hybridization (FISH), and interlaboratory reproducibility. Appl Immunohistochem Mol Morphol. 2007 15 94-102. 

Cheang MC, Treaba DO, Speers CH, et al. Immunohistochemi-cal detection using the new rabbit monoclonal antibody SPl of estrogen receptor in breast cancer is superior to mouse monoclonal antibody 1D5 in predicting survival. J Clin Oncol. 2006 24 5637-5644. 

As a result of the popularity of rabbit monoclonal antibodies, confusion exists when using the term monoclonal. Previously, monoclonal antibodies were always from mouse and so detection systems were always based on binding to mouse monoclonal antibodies. Now with the popularity of rabbit monoclonal antibodies, it is not possible to use the term monoclonal to identify the species of the antibody. 

Fischer, T., Nagel, F., Jacobs, S., Srnmm, R., Schulz, S. (2008). Reassessment ofCXCR4 chemokine receptor expression in human normal and neoplastic tissues using the novel rabbit monoclonal antibody UMB-2. PLoS One, 3. 

Miyake et al reported an ELISA method for the determination of pesticide residues in the aquatic environment. The polyclonal antibody and three monoclonal antibodies of acifluorfen were prepared by immunization of rabbits and mice with acifluorfen-bovine serum albumin conjugates. The polyclonal antibody reacted with acifluorfen at concentrations of 1.5-800 mg L , while the monoclonal antibodies reacted with acifluorfen at concentrations of 1.5-144 mg L . Among three monoclonal antibodies, AF 75-144 reacted with chlornitrofen, which did not react with the other two antibodies. It seems that the ELISA method is effective for the determination of herbicide residues in the aquatic environment. 

Sekido N, Mukaida N, Harada A, Nakanishi I, Watanabe I, Matsushima K. Prevention of lung perfusion injury in rabbits by a monoclonal antibody against interleukin-8. Nature 1993 365 654-657. 

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Many variations on the assay exist, but the ELISA, shown schematically below, is currently highly favored because of its simplicity once established in a laboratory sensitivity, detecting about one adduct per 107 bases and ability to screen many samples because of easy automations. The current prerequisite for the assay is that DNA can be modified to sufficiently high levels with the ultimate carcinogen to make it suitably antigenic. These types of antigens have been used to raise polyclonal antibodies in rabbits (41) and monoclonal antibodies from mice (42). 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

Caspase-3 Activation Analysis fixed cells were washed and incubated overnight with rabbit anti-active caspase-3 monoclonal antibody followed by FITC goat anti-rabbit antibody. Then cells were washed and mixed with PI and analyzed by BD FACSCalibur flow cytometer. For WB, cells are lysed with SDS and proteins were analyzed with caspase 3 Asp-175, p53 and yH2AX Ser-139. 

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