Antibodies, continued monoclonal

Generally, polyclonal antibodies are easier to produce, and high-affinity polyclonal antibodies can be obtained. Monoclonal antibodies are more specific to a certain epitope. They provide continuous production of exactly the same defined reagent and are more preferable for excess-reagent assays. The double sandwich technique has used two antibodies from monoclonals or combinations of mono- and polyclonals, with specificity against two different epitopes of the analyte. One antibody functions as a capturing antibody for the analyte and the other as the label carrier (118). 

Mammalian Cells Unlike microbial cells, mammalian cells do not continue to reproduce forever. Cancerous cells have lost this natural timing that leads to death after a few dozen generations and continue to multiply indefinitely. Hybridoma cells from the fusion of two mammalian lymphoid cells, one cancerous and the other normal, are important for mammalian cell culture. They produce monoclonal antibodies for research, for affinity methods for biological separations, and for analyses used in the diagnosis and treatment of some diseases. However, the frequency of fusion is low. If the unfused cells are not killed, the myelomas 1 overgrow the hybrid cells. The myelomas can be isolated when there is a defect in their production of enzymes involved in nucleotide synthesis. Mammahan cells can produce the necessary enzymes and thus so can the fused cells. When the cells are placed in a medium in which the enzymes are necessaiy for survival, the myelomas will not survive. The unfused normal cells will die because of their limited life span. Thus, after a period of time, the hybridomas will be the only cells left ahve. 

Commercial use of cell and tissue culture continues to expand. Improvement of organisms through recombinant nucleic acid techniques has become commonplace. Formerly, a few laboratories were well ahead of most others, but now the methods have been perfected for routine use. Another technique that is widely practiced is culturing of cells that excrete high concentrations of just one antibody protein. The specificity of antibodies and antigens is exploited in medical testing procedures using these pure monoclonal antibodies. 

In the dialyzed batch start-up phase and the subsequent continuous operation a substantial increase in viable cell density and monoclonal antibody (MAb) titer was observed compared to a conventional suspension culture. The raw data, profiles of the viable cell density, viability and monoclonal antibody titer during the batch start-up and the continuous operation with a dialysis flow rate of 5 L/d are shown in Figures 17.6 and 17.7. The raw data are also available in tabular form in the corresponding input file for the FORTRAN program on data smoothing for short cut methods provided with the enclosed CD. 

Figure 17.6 Dialyzed Chemostat Monoclonal antibody concentration (raw and smoothed measurements) during initial batch start-up and subsequent dialyzed continuous operation with a dialysis flow rate of 5 L/d. [reprinted from the Journal of Biotechnology Bioengineering with permission from J. Wiley],...

The objective of this exercise is to use the techniques developed in Section 7.3 of this book to determine the specific monoclonal antibody production rate (qiw) during the batch start-up and the subsequent continuous operation. 

At time t=212 h the continuous feeding was initiated at 5 L/d corresponding to a dilution rate of 0.45 d . Soon after continuous feeding started, a sharp increase in the viability was observed as a result of physically removing dead cells that had accumulated in the bioreactor. The viable cell density also increased as a result of the initiation of direct feeding. At time t 550 h a steady state appeared to have been reached as judged by the stability of the viable cell density and viability for a period of at least 4 days. Linardos et al. (1992) used the steady state measurements to analyze the dialyzed chemostat. Our objective here is to use the techniques developed in Chapter 7 to determine the specific monoclonal antibody production rate in the period 212 to 570 h where an oscillatory behavior of the MAb titer is observed and examine whether it differs from the value computed during the start-up phase. 

Linardos, T.I., N. Kalogerakis, L.A. Behie and L.R. Lamontagne, "Monoclonal Antibody Production in Dialyzed Continuous Suspension Culture", Biotechnol. Bioeng, 39, 504-510 (1992). 

Inhibition of cytokine activity in vivo by administration of monoclonal antibodies (and, more recently, by gene knockout studies) continues to elucidate the physiological and pathophysiological effect of various cytokines. 

Hybridoma Cell produced by the fusion of antibody-producing plasma cells with myeloma/carcinoma cells. The resultant hybrids have then the capacity to produce antibody (as determined by the properties of the plasma cells), and can be grown in continuous culture indefinitely owing to the immortality of the myeloma fusion partner. This technique enabled the first continuous supply of monoclonal antibodies to be produced. 

Somehow, Seebach ends his superb review, in which more than 500 references are quoted, with a rather optimistic message "that organic synthesis continues to react forcefully and with vitality to new challenges, still ready to pursue old dreams", and he refers to some exciting new targets such as supramolecular structures inhibitors, suicidal substrates and flustrates monoclonal antibodies and... 

Fig. 3. Immunofluorescence localization of tubulin in microtubules in Swiss 3T3 cultured fibroblasts. Swiss 3T3 cells were fixed in glutaraldehyde followed by treatment with sodium borohydride (4). Tubulin was detected using a rat monoclonal antitubulin antibody (YLl/2), followed by goat antirat IgG conjugated to rhodamine, all steps in the continuous presence of 0.1% saponin. Note the individual microtubules visible under the dark nuclear region (bar = 4 im).

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