DETERMINATION OF ORIENTATION OF THE IGG MOLECULES IN IMMOBILIZED LANGMUIR MONOLAYERS BY MEANS OF BINDING WITH FRAGMENT SPECIFIC ANTIIMMUNOGLOBULIN ANTIBODIES

We report the technique for determining the orientation of the Ige mol ecules in Langmuir films immobilized on a solid substrate. This techni que is based on measuring the binding of the secondary antibodies spec ifically recognising Fab and Fc fragments of immobilized IgG molecules . Specific binding of anti-Fab and anti-Fc rabbit anti-mouse (RAM) pol yclonal antibodies to the film of mouse IgG shows strong dependence on surface deposition pressure of the film indicating changes of molecul ar orientation occurring in the film. At high deposition pressures the amount of anti-Fab specific RAM antibodies bound with the mouse IgG f ilm is much greater than the anti-Fc specific RAM antibodies. This mea ns that IgG molecules of the film are preferentially oriented with the ir Fab fragments outside. The ratio of molecules oriented in this way to the opposing molecules is estimated to be 3.125. The technique can be considered rather general and suitable for similar studies of any p rotein 2-dimensional arrays since the antibodies to different parts or fragments of any protein can be obtained.