Monoclonal antibodies supernatant

Yokoyama, W. 1991b. Monoclonal antibody supernatant and ascites fluid. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 2.6.1-2.6.7. John Wiley Sons, New York. 

Clausen, A. M., Subramanian, A., and Carr, P. W., Purification of monoclonal antibodies from cell culture supernatants using a modified zirconia-based cation-exchange support, /. Chromatogr. A, 831, 63, 1999. 

The hybridoma cells are initially grown in a medium that will not maintain the growth of the cancer cells these therefore die, as do non-fused lymphocytes, leaving only the fused cells. As the hybridoma cells grow, the supernatant fluid is tested for the presence of antibodies. Those cultures producing the desired antibody are further cloned and either grown in bulk or as tumours in animals and the monoclonal antibodies harvested. 

To reduce unspecific binding, mix a 200-pl aliquot of the clear supernatant with 2 pi pre-immune serum or unspecific antibody and a further 200 pi aliquot with 50 pi precipitation aid. Rock at 0 °C for 1 h and spin at 1000 x g. Transfer the supernatant into a fresh container and fill it up to 1000 pi with Soln. A. Add 0.5 - 5 pi of the specific antiserum and monoclonal antibody, respectively, and incubate on ice for 1 h. Prepare a second sample containing pre-immune serum instead of antiserum. 

A. General description Abciximab is the Fab fragment of the chimeric human-murine monoclonal antibody 7E3. It binds to the glycoprotein (gp) Ilb/IIIa receptor of human platelets and inhibits platelet aggregation. The chimeric antibody is produced by continuous perfusion in mammahan cell-culture. The 48kDa F b fragment is purified from cell-culture supernatant. 

The success of immunoprecipitation depends on the affinity of the antibody for its antigen as well as for protein G or protein A. In general, while polyclonal antibodies are best, purified monoclonal antibodies (MAb), ascites fluid, or hybri-doma supernatant can also be used. 

The primary antibody can be in the form of a polyclonal antiserum or a monoclonal antibody produced either in a culture supernatant or in an ascitic fluid The concentration of the antibody in an ascitic fluid will be an order of magnitude greater than that in the culture supernatant, but the latter will be free of other immunoglobulins. 

Resuspend the pellet in 0.5 mL of antibody incubation buffer and 25 pL of monoclonal antibody. The dilution of monoclonal antibody will vary according to the preparation (see Notes 5 and 6) The monoclonal antibody used in the author s studies is derived directly from the supernatant from the antibody producing cell line Incubate the tubes for 1 h at room temperature with occasional mixing (see Note 7)... 

The dilution of the antibodies has to be determined empirically Polyclonal antibodies can usually be diluted between 1.50 and 1.250 Monoclonal antibodies in tissue culture supernatant may need to be concentrated by centrifuging a frozen sample m a microfuge for 10 min and discarding the top third of the solution. This solution (100 pL) can then have the other primary antibody diluted into it in place of PBS-BSA Monoclonal antibodies produced as ascites can usually be diluted between 1-200 and 1 1000. 

Fig. 5.1 Technique to produce monoclonal antibodies. After mice are injected with the antigen, the splenocytes are isolated and fused with myeloma cells. The fused cells are cultured (1) and the supernatants are assayed for the presence of desired antibodies (2). The cells from antibodypositive wells are cloned (3) and supernatants are tested for the desired antibodies (4) followed by expansion of the positive clones (5) and in vitro or in vivo propagation to produce the needed amount of monoclonal antibodies (see Color Insert)...

Monoclonal antibodies (MoAb) will be separated from the supernatant of a mammalian culture by using DEAE-Sephacel as the adsorbent. The supernatant contains 100 jtg/mL of MoAb. Adsorption follows the Langmuir isotherm with Y max =116 mg of MoAb per mL of adsorbent (settled volume) and K L = 0.5 x 1(T3 mg of MoAb per mL of supernatant (Desai et al, 1987). The amount of supernatant is 1 L. If you use 2 mL of DEAE-Sephacel for a single-stage, what percent of the MoAb will be recovered ... 

Malignant lesions and normal tissue biopsies (colon) are fixed with formalin and embedded in paraffin (Hernandez-Blazquez et al., 2000). Paraffin sections (4 p,m thick) are deparaffinized, rehydrated, and rinsed in PBS. They are placed in 0.1 mM citrate buffer (pH 3.4), heated for 10 min in a microwave oven at full power (720 W), and washed in PBS. The slides are immersed in 2N HC1 for 2 hr at 37°C and then rinsed in PBS. The sections are covered with 100 p,l ofhybridoma supernatant containing anti-5-MeCyd monoclonal antibody (5 (xg/ml) and incubated for 1 hr at room temperature with a biotinylated goat antimouse secondary antibody diluted 1 200 in PBS containing 0.1% BSA. 

Harvest the cells aseptically using a sterile syringe and needle and sediment them at 800g for 5 min and remove the supernatant (from which monoclonal antibodies may be prepared). 

In more practical terms, titers may vary from 1 100 to 1 2000 for polyclonal antisera, from 1 10 to 1 1,000 for monoclonal antibodies in cell culture supernatants, and up to 1 1,000,000 for monoclonal antibodies in ascites fluid. These dilutions may likely be... 

Lutkemeyer, D., Bretschneider, M., Buntemeyer, H., and Lehmann, J. (1993). Membrane chromatography for rapid purification of recombinant antithrombin III and monoclonal antibodies from cell culture supernatant. J. Chromatogr. 639, 57-66. 

Cell culture supernatants containing fetal bovine serum (from 1 to 10%) are characterized by their content of serum proteins and their low concentration of monoclonal antibodies (50-500 mg/mL). A perequisite for a good separation is to eliminate cells and cell debris first in some cases, the concentration of the feedstock is also a requirement. Special care must be taken for the choice of the fetal bovine serum so as it does not carry any trace... 

FIGURE 12 Purification of a murine monoclonal antibody against IgE form hybridoma culture supernatant on a phenyl Sepharose HP. The feedstock has been added with ammonium sulfate to a final concentration of 0.5 M. The sample volume was 130 mL. The column was operated at 100 cm / hr. As equilibrium buffer a 20 mM potassium phosphate buffer, pH 7.0, supplemented with 0.5 M ammonium sulfate was used. Elution was performed by a linear gradient with a 20 mM potassium phosphate buffer, pH 7.0. The gradient volume was equal to 10 column volumes. 

Thiophilic adsorption chromatography has been described for the purification of murine monoclonal antibodies from hybridoma cell culture containing fetal bovine serum.154 Due to the very low concentration of immunoglobulins in cell culture supernatants, binding capacity remains modest in spite of the presence of 0.5 to 1 M potassium sulfate. Further developments of this technology described by Nopper et al.155 indicated that thiophilic sorbents could be modified in their structure to increase the specificity and the binding capacity. 

Figure 18 shows a separation of IgGl from a cell culture supernatant. As shown by electrophoresis, the purity of monoclonal antibodies in a single pass is of about 80-95%. This method represents the most advanced separation method for immunoglobulin for its good compromise between selectivity, binding capacity, and cost. 

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