Immunoadsorbent polymeric material and method of making same

#This invention relates to immunoassay and competitive protein binding procedures and to a device for use in such procedures and pertains more specifically to a method and apparatus for radioimmunoassay, enzyme immunoassay, and fluoroimmunoassay of antigens.

#It has previously been proposed in Catt U.S. Pat. No. 3,646,346 to provide a solid water-insoluble polymeric material, preferably in the form of a test tube, having its surface coated by adsorption of an antibody to the protein to be assayed, the coated tube then being brought into contact with an aqueous sample containing the protein to be assayed and also with a known quantity of the same protein which is radioactively labeled, finally separating the polymeric surface from the aqueous sample and determining, by measuring the emitted radiation, the amount of labeled protein which has been bound to the coated polymer. The solid phase antibody coated polymeric material can be made and used in the Catt procedure only when the antiserum containing the antibody to be adsorbed is of moderately high titer; moreover, the coated polymeric material and procedure cannot be used with some binding proteins such as hog gastric intrinsic factor (a binding protein for vitamin B-12) which are not adsorbed on polymeric surfaces sufficiently even when available in high titer solutions.

#It has been proposed by Avrameas et al., Immunochemistry, Vol. 6, pages 53-66 (Pergamon Press, 1969) to insolubilize antigens or antibodies by cross-linking them with glutaraldehyde and to employ the insolubilized proteins thus produced, either in column procedure or in batchwise operation, for the isolation of antigens or antibodies. It has also been proposed by Avrameas, loc. cit., pages 43-52 to couple enzymes to human immmunoglobulin-G and serum albumin as well as to sheep and rabbit antibody with glutaraldehyde and to use the products, which retain a substantial part of their immunological and enzymatic activity, for the intracellular detection of antigens and antibodies and for characterization of antibodies after immunoelectrophoresis. It has in addition been proposed by Barrett et al. at the Ninth International Congress on Clinical Chemistry, 13-18 July, 1975, Toronto, to bring a plastic tube into contact with glutaraldehyde to provide reaction aldehyde residues on the surface of the plastic, and then to bring the plastic into contact with antibody to cause the antibody to be coupled to the glutaraldehyde.

#The gamma globulin fraction of a rabbit antiserum to thyroxine (tetraiodothyronine, T4) is isolated by treating one ml. of the serum for 18 hours at 4.degree. C. with an equal volume of 80% saturated ammonium sulfate, pH 7.1. The precipitated gamma globulin fraction is spun down in a refrigerated centrifuge at 3000 rpm. for 30 minutes, then diluted in 2000 ml. of an isotonic 0.01M phosphate buffer pH 6.0. To this is added 400 ml. of 0.02M glutaraldehyde cross-linking reagent in distilled water. This mixture is allowed to react for 20 minutes at 37.degree. C. and is then diluted for coating by the addition of 2000 ml. of 1M phosphate buffer pH 8.5., to give a final dilution of cross-linked gamma globulin of 1/4400. One ml. aliquots of this coating solution are introduced into the bottom of clean, dry polypropylene plastic tubes. After incubation for 1 hour at 37.degree. C., the tubes are aspirated and washed twice with an isotonic 0.01M tris buffer pH 7.4 containing 0.1% gelatin to remove any unadsorbed cross-linked gamma globulin. The tubes are dried at ambient temperature (22.degree.-25.degree. C.) and stored in sealed plastic bags.

#A series of assays was run in these coated tubes using 1 ml. of an isotonic 0.01M tris buffer pH 7.4 containing 0.1% gelatin, quantities of inhibitor T4 ranging from 41 pg to 10 ng per tube and 14,000 cpm of .sup.125 I labeled T4 as radiolabeled tracer followed by room temperature incubation for 3 hours. At the minimum quantity of added T4, 59% of the added tracer was bound to the coated tube, decreasing amounts of tracer being bound at higher levels of added T4 as shown in the drawing.

#Analysis of the data by a modification of the procedure of Pinckard et al., Handbook of Experimental Immunology, page 498 (Great Britain 1967) gives an apparent affinity constant (K) of 4.5 .times. 10.sup.9 M.sup.-1 and the amount of T4 antibody bound to the tube (Ab.sub.o) of 1.8 .times. 10.sup.-10 M.