Antibody production, technique

Both of us have learnt a lot about new antibody production techniques while editing this book and we hope this will be the same experience for all whom refer to the finished volume. 

Crane.L. (1987) In Monoclonal Antibody Production Techniques and Applications. Schook,L.B. (ed.), Marcel Dekker, Inc., New York, Chapter 9. 

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. 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

Immunohistochemistry of multiple antigens in monolayer cell cultures during the same experimental conditions is also important in cell biology. Advanced techniques for antibody production combined with sensitive detection systems have facilitated the localization of... 

A number of non-hybridoma techniques have been developed for in vitro antibody production a review of recombinant antibody production is covered in Chapter 6. 

Marco, M.P. and B.D. Hammock (1995). Immunochemical techniques for environmental analysis. II. Antibody production and immunoassay development. Trends Anal. Chem., 14 415 125. 

The need for a successful large-scale mAb production technique is indicated by the growing commercial market for antibody-based products and the increased importance of in vivo diagnostic as well as therapeutic applications. According to market research, total sales of in vivo and in vitro mAb-based products reached approximately 8 billion in 1993 and this volume is expected to increase in subsequent years. Therefore, significant commercial production of mAbs is being emphasized. In one procedure, mAb-producing murine hybridomas are cultured in 40-liter bioreactors. 

A number of immunological techniques have been used in comparative studies.8,9 The most important of these is microcomplement fixation (MC F), a quantitative technique that has played a key role in many classic studies of molecular evolution and molecular systematics. By selecting proteins with different rates of evolution, a broad range of divergences can be examined. The cost of the technique is moderate, but biochemical expertise is required and the labor involved is substantial. Protein must be purified from some or all taxa for antibody production, and, for those taxa, a sizable tissue or serum sample is needed. Antibody production itself is usually done in rabbits, so an animal care facility must be available. Like isozyme electrophoresis, the large body of immunological distance data already available ensures the continued value of this technique for certain investigations. 

Hoffman et al.52, presented evidence that a single oral dose of tilorone enhanced the primary immune response to sheep red blood cells (SRBC) in mice as measured by the Jerne Plaque technique. They also reported an increase in hemolysin titer after tilorone administration. To further evaluate the action of tilorone on humoral antibody responses, Megel ef a/.3 have studied its effect on 19S and 7S production in the primary and secondary immune responses in mice. It was found that tilorone elevated 19S antibody titer on days 3 and 4 after immunization. After 9 days of continuous drug administration, the 19 S response for both groups was diminished compared to days 3 and 4 however tilorone was found to cause a significant increase in the 7S antibody production compared to controls. Tilorone also stimulated the 19S response to E. coli endotoxin, a thymus-independent antigen, on days 3 and 4 after immunization. 

A detailed discussion of antibody production and its control is beyond our purpose. However, minimal appreciation of the events leading to antibody production and a few definitions are necessary if immunochemical techniques are to be used knowledgeably. An antigen may be defined as any compound that (1) can stimulate production of antibodies when injected into a test animal and (2) reacts specifically with the antibodies produced. Both parts of this definition are necessary to distinguish antigens from haptens (see Figure 8-3), which are small molecules that... 

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