Conjugated monoclonal antibodies

Although many applications of flow cytometry involve the staining of cells for proteins expressed on the outer membrane, cells also have many proteins that are not displayed on their surface. With appropriate procedures, flow cytometry can provide a means to analyze these intracellular proteins. The outer cell membrane is impermeable to large molcules like antibodies however, if we intentionally fix cells to stabilize proteins and then disrupt the outer membrane, the cells can be stained with fluorochrome-conjugated monoclonal antibodies against intracellular proteins. After time to allow the antibodies to pass through the now-permeabilized membrane, the cells are washed to remove loosely bound antibodies and then are run through the flow cytometer to measure their fluorescence intensity. 

Cytotoxicity may decrease cell surface expression and transcription of markers. Thus, if concentration of test chemical induces low cell viability then measurement of cell surface expression and transcription is limited. In addition, donor variability may introduce differences in fluorochrome-conjugated monoclonal antibody expression (Aiba et al. 1997). 

The fluorochrome-conjugated monoclonal antibodies used may be varied. Investigation on one, a combination or all may be performed. In addition RNA Isolation to confirm the quality of RNA and Genochip Microarray for transcript profiling may be performed after flow cytometry evaluation. 

Fully human antibodies can be constructed from phage libraries. In addition to the diversity of engineered antibodies, other molecules can be attached to the antibody, such as enzymes, toxins, viruses, radionuclides, and biosensors for targeting, imaging, or diagnosing. These are commonly referred to as conjugated" monoclonal antibodies. 

Conjugated monoclonal antibodies can be used as carriers of toxic therapy, such as radionuclides, (e.g., yttrium-90 ibritumomab tiuxetan and iodine-131 tositumomab), cytotoxic drugs, or cell toxins to specific cell targets. They are also being employed to create tumor vaccines by stimulating a host antibody reaction causing the production of anti-idiotype antibodies. 

FITC-conjugated monoclonal antibody (MAb) against an intracellular viral antigen. In the case of HIV-1 a specific anti-p24 MAb, KC57, is available from Coulter Electronics Ltd. (UK). 

Mass cytometry, however, has important limitations. First of all, the cells employed in the analysis cannot be recovered. Second, the potential huge number of labels complicates the analysis and data interpretation. Finally, the prize of the stable isotope conjugated monoclonal antibodies is quite high, also necessary to test their specificity, affinity, stability, and resistance (43). 

Cryptosporidium oocysts were selectively extracted (antibodies coupled to magnetic beads) from water concentrates using IMS. These oocysts were subsequently visualized using FITC conjugated monoclonal antibodies. However, dissociation from the immunomagnetic beads before labelling proved to be necessary (de Roubin et al. 2(X)2 Reynolds et al. 1999 Rushton et al. 2000). 

Conjugates of Acridinium and MAb Anti-HCV core. Acr-cll-10 IgG (Control Conjugate). Monoclonal antibody (MAb) cl 1-10 anti-HCV core IgG was directly labeled with N-hydroxysuccinimide ester of N-sulfopropyl N-tosylamide acridinium (Fig 1) in a phosphate buffer. After conjugation, the conjugate was separated by sec-HPLC into fractions with Acr/IgG molar ratios ranging from 3 to 5 estimated by A370 and A28o-... 

J.K. Steele, D. Liu, A.T. Stammers, S. Whitney, J.G. Levy (1988). Suppressor deletion therapy selective elimination of T suppressor cells in vivo using a hematoporphyrin conjugated monoclonal antibody permits animals to reject syngeneic tumor cells. Cancer Immunol. Immunother., 26, 125-131. 

Lewis M R, Kao J Y, Anderson A-L J, et al. (2001). An improved method for conjugating monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA. Bioconjug. Chem. 12 320-324. 

Table 4 Staining for surf e and intracellular antigens with ready-conjugated monoclonal antibodies (mAbs) before and after fixation ...

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