The use of immunoglobulin/antigen interactions as the basis for assuring specificity in quantitative assays has now been practiced for more than two decades. The literature describing a variety of such assays is extensive, and the number of possible protocols is almost prohibitive of a list which is all inclusive. The particular methods to which this invention is directed involve formation of a labeled immune complex or other specific complex and solubilizing this complex. There are a large number of protocols for even this limited approach, which, however, have some features in common.
Of course, at least one component involved in the assay must be, or become, detectable in some manner. In this majority of cases, this means that one of the participants in the specific reaction such as an immunoreaction must be labeled in some fashion. Various labels have been employed: radioactive isotopes, fluorophores, colored substances, enzymes (which can then be used to catalyze reactions with detectable products of reactants) and even macroscopic particles which permit visualization. The most sensitive of these assays utilize enzymes, radioactive substances, or fluorophores.
In all these procedures, the quantity of label measured must be a function of the quantity of analyte present in the sample. This can be arranged, basically, in two ways: either the labeled component is caused to react directly or indirectly with the analyte in the sample, or the analyte in the sample is allowed to compete with the component carrying the label for a substance specifically reactive with the analyte.
In most protocols, a physical separation must be effected between the affinity partners which have reacted by virtue of the specific reaction and those that have not. Most frequently, the immune or other complexes formed are either precipitated or are adsorbed to a solid support. If precipitated, the complexes are generally harvested by centrifugation; if adsorbed to a solid support, the complexes are separated from nonreactive materials by washing with solutions which do not react with adsorbent. In most assay designs, the label which is used for quantitation is included in and measured in the solid phase. However, in the assays to which this invention is addressed, the specifically formed complex is redissolved before measuring the label.
One such embodiment is described in U.S. Pat. No. 4,548,908 wherein the entire precipitated immune complex containing a fluorophore is dissolved in base for reading with a fluorometer. Since the base dissolves the entire complex, and does not separate the fluorophores from the complex, the fluorophores may be close enough to each other to quench, thereby reducing the total radiation potentially detectable from more dilute fluorophores (see, e.g., Smith, D. S., et al, Ann Clin Biochem (1981) 18: 253-274). Thus, there is a limitation on the amount of detectable emitted light due to the proximity of the fluorophores to each other even on the solubilized proteins. This problem arises also for alternative forms of labeling, in that, for example, enzyme activity may be damped by the protein to which it is conjugated and (less seriously) radiation may be absorbed by the complex to which the radioisotope is bound.
It would be helpful in increasing sensitivity and accuracy if the fluorophore or other label could be individually resolubilized and detached from the protein or other affinity partner to which it is conjugated, whether or not the specific complex is itself resolubilized. Were this achievable, the fluorophore molecules or other label would behave as independent moieties so that they have increased detectability, i.e., reduced capacity to quench, or otherwise alter, their detectability.
U.S. Pat. No. 4,231,999 to Carlsson et al discloses the use of a splittable bond in the conjugation of a label to a component of an immunoassay which permits the label to be detached for measurement. The label is detached directly from the solid support, and is freed for the purpose of overcoming difficulties in measurement of label on solid materials. For fluorescent-labeled assays which involve reading emitted light where the fluorescent complex is adsorbed to a solid support, for example, the high concentration of fluorophore on the solid results in too high a level of light for accurate measurement (akin to attempting to view the stars in the daytime). To solve this problem of high emission a number of suggested splittable bonds are disclosed, including disulfide linkages. U.S. Pat. No. 3,232,119, also to Carlsson et al, specifically outlines preferred procedures for preparing labeled moieties conjugated through disulfide linkages which are cleavable through reduction. Again, the immune complex formed is directly cleaved by reduction prior to measurement of label.
The Carlsson disclosures suffer from the disadvantage that the sensitivity of the assay is limited by the availability of cleavable bonds at the surface of the immune complex. The reaction must either be conducted as a monolayer, or a loss of accuracy will result from failure to cleave the bonds of the conjugate which are internal to the bulk complex. The invention herein solves these problems by utilizing a presolubilization step prior to detachment of label.