Chemistry: analytical and immunological testing – For preexisting immune complex or auto-immune disease
Patent
1981-04-20
1984-06-05
Marantz, Sidney
Chemistry: analytical and immunological testing
For preexisting immune complex or auto-immune disease
204403, 435 7, 436516, 436530, G01N 3354, G01N 3368, G01N 2726
Patent
active
044529015
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns new solid supports for proteins for analytical purposes, a process for their preparation, and their use, especially as diagnostic tools.
More particularly the invention is directed to porous nitrocellulose sheets containing a replica of an electropherogram of proteins in a gel. The form in which the proteins are immobilized on the nitrocellulose sheet is excellently suitable for analytical purposes, such as the detection and identification of proteins by solid state immuno-assay methods, such as those currently used, for instance radioactive methods, fluorometric methods or enzyme immuno-assays.
The invention thus comprises also the use of the new solid supports for proteins for the immuno detection of proteins, antigens and antibodies, especially as diagnostic tools.
The invention is chiefly based on the finding that it is possible to transfer proteins from a gel (as currently used in protein biochemistry, e.g. a gel used for the electrophoretic separation of proteins) on to sheets of nitrocellulose, that is to say on cellulose nitric acid ester supports in the form of thin sheets, which may contain in addition other cellulose esters, such as cellulose acetate. The transfer of the proteins to the gel can be quantitative, and the immobilized proteins form an exact replica of the pattern which was present in the gel.
Polyacrylamide gel electrophoresis has become a standard tool in every laboratory in which proteins are analyzed and purified. Most frequently, the amount and location of the protein are of interest and staining is then sufficient. However, it may also be important to correlate an activity of a protein with a particular band on the gel. Enzymatic and binding activities can sometimes be detected in situ by letting substrates or ligands diffuse into the gel. In immunoelectrophoresis, the antigen is allowed to diffuse or electrophoretically move against antibody. A precipitate is then formed where the antigen and antibody interact. Modifications have been described in which the antigen is precipitated by directly soaking the separation matrix in antiserum. The choice of electrophoretic systems is then severely limited by the need to have a gel of sufficiently large pore size as to permit the diffusion of the antibody and/or antigen. Such systems are also dependent on concentration and type of antigen or antibody to give a physically immobile aggregate. Attempts have therefore been made to transfer the proteins isolated in gels on to solid supports, as will be described below.
The transfer of the proteins from the gel to the nitrocellulose support is carried out, according to the present invention, by an electric field applied to the gel containing the proteins, causing an electrophoretic migration of the latter, for instance previously separated by some standard electrophoresis procedure, and in the form of an electropherogram. The electric field is applied so that the proteins will migrate in the direction of the nitrocellulose sheet which is in contact with the gel, preferably perpendicular to the plane of the gel. As is explained below, it is surprising that it is possible to obtain a faithful replica of the array of the proteins present in the gel, because spreading of the proteins in the gel under the influence of the inhomogeneous electric field would have been expected, as well as unpredictable complications due to the presence of ionic detergents and because the chemical basis for the binding of the proteins to nitrocellulose is not understood. The immobilized proteins on the nitrocellulose sheet are stable against washing the sheet, for instance against treatment with suitable salt solutions, for instance saline (physiological sodium chloride solution).
If the sheets are to be used for diagnostic purposes or other scientific experiments involving immuno-assays procedures, the sheets shall be treated with appropriate proteins which will satuate the residual adsorption capacities of the nitrocellulose sheet. This is done by saturating the sheet with an individ
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Gordon Julian
Staehelin Theophil
Towbin Harry
Ciba-Geigy Corporation
Feit Irving N.
Marantz Sidney
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