The present invention relates generally to the immobilization of proteins in the presence of alkyl sulfate detergents using high concentrations of renaturing surfactants, and immuno-assays using such immobilized proteins. A related application, Ser. No. 07/346,108, is filed concurrently herewith.
In recent years, enzyme immuno-assay procedures have come into wide spread use in the diagnosis of various disorders and conditions of the human body. Such techniques, commonly referred to as EIA, make use of the mechanisms of immune systems in higher organisms. Antibodies are produced in response to the presence in the organisms of antigens which are pathogenic or foreign to the organisms. For example, the human body may produce antibodies in response to and which are capable of reacting with a particular antigen, and it is that interaction which forms the basis for many EIA techniques.
As is now well understood, antigens are typically high molecular weight proteins which have any of a variety of functional chemical groups. Antibodies are proteins produced by the immune system of the human body which have the capability of reacting specifically with those antigens. One such EIA technique which can be used to determine the presence of antibodies in body fluids is the EIA technique described in European Patent Publication No. 0 217 403 A2 published on Apr. 8, 1987. In the immuno-assays described therein, microparticles which contain reactive functional groups react with an antigen to immobilize the antigen on the surfaces of the microparticles. The immobilized antigen is then contacted with, for example, a body fluid which is suspected of containing antibodies to the antigen. The antibodies, having a specific reactivity with the immobilized antigen, become chemically bonded to the antigen which has been immobilized. The matrix is then contacted with a second reactant which contains a label having the capability of reacting chemically with the antibodies bound to the immobilized antigen, to produce a detectable response indicative of the presence and/or amount of the antibodies contained in the sample of body fluid.
Thus, the EIA technique as described in that European Patent Publication depends on the immobilization of the antigen without impairing its reactivity to specific antibodies for which a response is desired. Of course, it is also possible to immobilize antibodies specific for a desired antigenic analyte.
Many proteins or antigens which are used in such EIA techniques become insoluble as a result of the formation of aggregates among them. For that reason, such protein reagents must be used in systems in which the proteins are solubilized so that they can be immobilized on the microparticles. It has become conventional to solubilize such proteins using a surfactant such as urea, guanidine hydrochloride or alkyl sulfate detergents such as sodium dodecyl sulfate (SDS), to maintain the stability of protein in the solution so that it can be immobilized on the surface of the microparticles.
It is known that the use of SDS, while effective in promoting the solubility of antigenic proteins, can also produce problems. Detergents like SDS are often referred to as denaturing surfactants, causing the protein to unfold and thereby lose its reactivity. In systems where it is desired to covalently immobilize the protein on a solid phase test medium as is described in the aforementioned European patent publication, the denaturing effect of SDS or other alkyl sulfate detergents may result in an inactive or non-reactive protein being bound to the solid phase.
It has been proposed to replace SDS by using high concentrations of urea or guanidine hydrochloride. However, that approach has been found to produce an increase in the non-specific reactivity of the protein in various immuno-assays.
Immuno-assays are now widely used for the detection of proteins, and particularly for detecting the presence of antibodies and antigens in a given system. One of the immuno-assays which is finding ever-increasing widespread use is the assay for detecting antibodies to HIV. Several antigens have been developed, some through genetic engineering techniques, for use in detecting antibodies to HIV. Some of those are described in co-pending and co-owned application Ser. No. 020,282, filed Feb. 27, 1987, no abandoned, and entitled "Immuno-Assays for Antigens to HIV." Antigens used in detecting antibodies to HIV have been found to be either insoluble or poorly soluble in aqueous solution, and those solubility characteristics have prevented or impaired the immobilization of those antigens on microparticles of the kind described in the foregoing European patent publication.
The HIV antigens discussed are, like many other antigens known in the art, generally solubilized with alkyl sulfate detergents, and particularly SDS. It has been found, however, that when antigens solubilized with SDS are immobilized, the antigen proteins are at the same time denatured, rendering the immuno-assays using them ineffective. It has been found that such antigens not only lose reactivity, they at the same time lose their specificity for reaction with the antibodies found in body fluids. Thus, it has become necessary to develop a system which is capable of not only solubilizing such antigens, and other proteins requiring the presence of alkyl sulfates for solubility, but also ensuring that the specific reactivity of those antigens is retained.
Various other techniques have been used to renature, or to prevent denaturation of, proteins generally. For example, betaines have been used to prevent denaturation of enzymes solubilized with alkyl sulfates. Further, it was found that denatured enzymes could be renatured after addition of betaines and subsequent passage of the solution through an ion exchange resin to remove the alkyl sulfates. Another previously used technique employed electrophoresis to remove alkyl sulfates, resulting in the renaturation of proteins solubilized with the alkyl sulfates. Such methods, however, are not conventionally used and are impractically employed in a commercial setting as a result of the specialized and prohibitively expensive equipment employed in such techniques and the large amount of time required by those methods.