Methods for obtaining monoclonal antibodies useful in enhanced sensitivity immunoassays

#Development of the hybridoma technology [Kohler, G., and C. Milstein, (1975) Nature 256: 495 and; Goding, J. W., (1980) Immunological Methods 39: 285] has provided immunoglobulin reagents which bind to only one antigenic site. Although these reagents have found widespread use as biochemical and immunological tools, their usefulness in radioimmunoassay has frequently been limited by their lower affinity for antigen compared with that of serum antibodies [Goding, J. W., (1980) Immunological Methods 39: 285]. In principle, the affinity of monoclonal antibodies could be enhanced by more stringent hybridoma selection procedures. The production of monoclonal antibodies has also enabled investigators to dissect the humoral immune response into its pure components [Staines, N. A. and A. M. Lew (1980), Immunology 40: 287]. This will eventually result in a more comprehensive understanding of the role of the individual antibody, especially with regard to the possibility that an antiserum may have different characteristics than the sum of its individual antibodies.

#During the course of a systematic assessment of the immunochemistry of human chorionic gonadotropin (hCG), including the relative orientation of different epitopes of this molecule and the effect of several monoclonal antibodies on the hormone-receptor interaction, it has been observed that the apparent affinity of mixtures of certain monoclonal antibodies is enhanced relative to the affinity of the individual antibodies.

#A mixture of monoclonal antibodies which includes an effective assaying amount of each of at least two monoclonal antibodies that bind to different antigenic sites on an antigen and are capable of doing so under appropriate conditions is useful in an enhanced sensitivity assay for the antigen.

#FIG. 1: Radioimmunoassay for human chorionic gonadotropin (hCG) by double antibody liquid phase assay. The amount of labeled hCG bound in the absence of unlabeled inhibitor was approximately the same for each antibody or antibody mixture. This was achieved by appropriate dilution of each antibody solution or mixture. Rabbit anti-mouse IgG was the second antibody. The ED.sub.50 plus/minus the standard deviation was calculated for each antibody or mixture. The ED.sub.50 for the mixture of B101 and B102 is 2.44.+-.0.74.times.10.sup.-10 M, antibody B101 is 2.88.+-.0.74.times.10.sup.-9 M, and antibody B102 is 1.058.+-.0.995.times.10.sup.-7 M.

#FIG. 2: Scatchard analysis of the binding to hCG of A, a mixture of antibodies B101 and B102; B, antibody B101 and C, antibody B102. Slopes of the lines (therefore, the equilibrium binding constants) are A: 5.4.times.10.sup.9 ; B: 5.1.times.10.sup.8 ; and C: 1.9.times.10.sup.7.

#FIG. 3A: Radioimmunoassay for hCG by double antibody liquid phase assay. The procedure was the same as for FIG. 1. The ED.sub.50 for antibody A102 is 8.52.+-.2.94.times.10.sup.-9 M, antibody B101 is 2.88.+-.0.74.times.10.sup.-9 M, and mixture of antibodies B101 and A102 is 4.66.+-.1.99.times.10.sup.-9 M.

#It has been observed that mixing monoclonal antibodies directed against various epitopes of human chorionic gonadotropin can increase the sensitivity of antigen binding assays. Depending on the antibody pair chosen, the affinity of the mixture can be as much as 10-fold higher than that of the monoclonal antibodies assayed separately. This increased affinity can be detected in both a solid phase assay and a liquid phase double antibody radioimmunoassay. The mechanism for the increase in affinity depends on the formation of a multi-component complex. Mixing two antibodies which cannot bind simultaneously does not result in enhanced affinity, but combining pairs which can bind at the same time under appropriate conditions results in higher sensitivity in an antigen binding assay. If one of the antibodies of a pair which results in enhanced affinity upon mixing is replaced by its F(ab) fragment, the enhancement is no longer detectable, indicating that it is unlikely that the enhancement is due to an allosteric effect. While the F(ab').sub.2 fragment shows some enhancement when mixed with another antibody, it is not as effective as the intact antibody.

#Based upon these discoveries and observations, the present invention provides mixtures of monoclonal antibodies useful in an enhanced sensitivity assay for an antigen. The antibodies useful in the practices of the inventions are also characterized by the fact that they are capable of binding to the antigen both under normal nondenaturing conditions and under conditions described hereinafter where antibodies otherwise capable of binding to the antigen are unable to do so.

#The invention described herein may in principle involve more than two monoclonal antibodies. Mixtures containing three, four or more antibodies each of which binds to a different antigenic site on the antigen may be particularly useful in assays for large molecules such as polypeptides having numerous subunits. However, for illustrative purposes the invention will be described by reference to mixtures which contain two such antibodies.