Evaluation of labelled monoclonal antibodies

Alkaline phosphate substrate solution 2-nitrophenyl phosphate (10 mg) in 10 ml of 10 mM diethanolamine buffer pH 9.5,0.5 

Peroxidase substrate solution 3,3, 5,5 -tetramethylbenzidine (100 jxl of a 10 mgftnl soluticHi in DMSO) and 7.5 (xl of 6% hydrogen peroxide (freshly prepared from 30% stock solution) in 10 ml of 0.1 M sodium acetate/dtrate buffer pH 6.0 Alkaline phosphatase stopping solution 2 MNaOH 

All volumes are 200 p. and all incubations are at 37 °C unless stated otherwise. Controls should include no antigen as well as no antibody . 

Coat the wells of the microtitre plate with antigen at 10 pg/ml in coating buffer and incubate for 3 h or at 4 C overnight. 

Remove the contents of the wells ty inversion and tapping the plate on paper towelling. Add blocking solution and incubate for 1 h. 

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Milenic D E, Roselli M, Mirzadeh S, et al. (2001). In vivo evaluation of bismuth-labeled monoclonal antibody comparing DTPA-derived bifunctional chelates. Cancer Biother. Radiopharm. 16 133-146. 

This chapter provides protocols for the most convenient methods of labelling mouse IgG monoclonal antibodies with enzymes (alkaline phosphatase and peroxidase), a fluorescent molecule (fluorescein), biotin, and DIG. A general protocol for the evaluation of the labelled monoclonal antibody is also given. 

Kobayashi, H., Wu, C., Kim, M.K., Paik, C.H., Carrasquillo, J.A., and Brechbiel, M.W. (1999) Evaluation of the in vivo biodistribution of indium-111 and yttrium-88 labeled dendrimer-1 B4M-DTPA and its conjugation with anti-Tac monoclonal antibody. Bioconjug. Chem. 10, 103-111. 

It is entirely possible that surface staining cannot be accomplished before fixation. Some antibody-antigen complexes cannot withstand chemical fixation and/or permeabilization. An empirical evaluation must be made. In this example, cells are first stained with a monoclonal antibody against a cell-surface receptor, fixed with ethanol, and then the DNA is stained with propidium iodide. The cells are analyzed for two-color fluorescence, the green of the fluorescein-labeled surface marker and the red of the labeled DNA intercalator. This approach works for antibody-antigens that are unaffected by fixation. 

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