Screening hybridomas

The two ELISA techniques described below have complementary applications. 

The direct binding assay is used for screening hybridoma supernatants and for initial characterization of antibodies. The only DNA antigen involved in this method is that which becomes absorbed to the surface of the wells. The quantity of this DNA is not known accurately but is probably only a few nanograms per well. Although this technique has been used to estimate levels of modifications in samples of DNA it suffers several drawbacks as a method for determining clinically relevant adduct levels. 

The second ELISA technique described here is the competitive assay. In this assay, the wells are coated with a uniform quantity of modified DNA and a dilute solution of the antibody is mixed with various quantities of the DNA samples that are to be analysed. When these mixtures are added to the coated assay wells, there is a competition for a limited amount of antibody between two classes of antigen. The more adducts on the DNA in solution, the greater is the tendency for the antibody to bind to this DNA at the expense of its binding to the DNA absorbed to the plastic surface. The assay is calibrated by use of standard concentrations of dissolved competing DNA carrying known levels of adducts. In this method  

For use in screening of hybridomas, the level of modification of the DNA used to coat the plates will depend upon the specificity of the antibodies sought, the heterogeneity of modifications present on the DNA, and the relative abundance of the modification in question. For screening hybridomas to obtain antibodies recognizing melphalan and cisplatin adducts, base modification fi equendes of 0.01 and 0.06 respectively proved to be satisfactory (9,18). 

DNA modified with drug to a relatively high level this should be pure DNA (e.g. highly purified calf thymus DNA from Merck) with a base modification frequency of approx. 0.01-0.06, stored at -80 °C 

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Willingham, M. C. (1990) Immunocytochemical methods usefnl and informative tools for screening hybridomas and evalnating antigen expression. FOCUS... 

Unlike their murine counterparts, rat antibodies bind poorly, if at all, to protein A, but some isotypes will bind to protein G. For this reason, we use a sheep antirat F[ab ]2 as a general reagent both for immunoprecipitations and for screening hybridoma supernatants. 

Lane, D. P. and Lane, E. B. (1981) A rapid antibody assay system for screening hybridoma cultures. J. Immunol. Methods 47, 303—307. 

Test wells exhibiting growth for the presence of monoclonal antibodies when the hybridoma growth covers approx 25% of the base of the cell well. The assay system used should mimic the final test format required. Commonly, Triple Antibody Sandwich (TAS) (5) enzyme-linked immunosorbent assay (ELISA) is use for screening hybridomas for specific antibody. It is important when testing hybri-... 

To screen hybridoma supernatants discard the blocking buffer and wash plates twice with 200 pL PBS-Tween per well. 

Bakkali L, Guillou R, Gonzague M, Q uciere C (1994) A rapid and sensitive chemiluminescence dot-immunobinding assay for screening hybridoma supernatants. J Immunol Metliods 170 177—184. 

Screen hybridoma supernatants by immunofluorescence assay with infected erythrocytes (see Note 16). 

Willingham MC (1990) Immunocytochcmical methods useful and informative tools for screening hybridomas and evaluating antigen expression. Focus 12 62-67... 

Buchanan D, Kamarck M, Ruddle NH (1981) Development of a protein A enzyme immunoassay for use in screening hybridomas. J Immunol Methods 42 179-185... 

Screening hybridoma culture supernatants for specific antibody... 

Monoclonal antibody undiluted cultare supernatant r en screening hybridomas, or culture supernatant diluted in PBS containing 1% (w/v) BSA and 0.1% (v/v) Twear20... 

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