Kinetic assay

#The present invention relates to small size haptens, more particularly to their conjugates with other large molecules as immobilised or mobile immunoreactants and their use in rapid assays, particularly immunoassays, especially for lateral flow tests or strip tests, flow through formats and flow immunosensors.

#Rapid lateral flow strip tests or flow immunosensors have many benefits including a user-friendly format, a relatively short processing time before receiving a test result in comparison with typical ELISA assays, and long-term stability. They are also relatively inexpensive to make.

#For the immunoassay of small size haptens, such as steroids or drugs, a so-called competitive technique is usually required. As coating antigens, haptens are typically either coated onto a solid phase such as a strip membrane as a capture dot, or a line, to react with dye-, enzyme- or particle-conjugated antibodies; or labelled with dye, or other chromatically-active particles and used as mobile immunoreactants to form a colour dot or band an immunocomplex with immobilised antibodies on the solid phase. In both configurations, the coated antigens, either immobilised on the solid phase or as mobile immunoreactants, compete with free sample, antigens for a limited number of antibody binding sites. Therefore, the hapten/antibody binding capacity in the capture line is an important criteria that determines the detectable hapten concentration range and hence sensitivity of the immunoassays.

#While the present invention is broadly as defined above, it will be appreciated by those persons skilled in the art that it is not limited thereto but that it also includes embodiments of which the following description provides examples. In addition, a better understanding of the invention will be gained through reference to the accompanying drawings in which:

#FIG. 1 shows the synthesis of 4-progesterone derivatives with extended length linkers.

#FIG. 2 shows the structures of one 7α-progesterone-OVA conjugate [(2)-OVA] and three 4-progesterone-OVA conjugates [(3)OVA, (5)-OVA and (7)OVA] with various length linkers (4-, 11-, or 18-atom linker).

#In the present disclosure, a “rapid assay” is characterised by the assay components, when mixed, having a reaction that does not require it to approach a state of equilibrium before a meaningful result may be obtained from the assay. The invention is particularly suited to a rapid assay where the assay component reactions do not approach equilibrium during the period that the assay is run. Such rapid assays are non-equilibrium, kinetic-based assays. Typically, the assay should take less than 20 minutes to perform, more suitably less than 10 minutes.

#A “large group” is a group of sufficient size to cause significant steric hindrance with respect to the binding of an attached hapten to a ligand when a linker of less than 10 atoms in length is interposed between the ligand and the hapten. Typically, in the present invention, a large group is a protein or polypeptide. Alternatively, the large group is an indicator, such as a fluorescent dye, for example bilirubin.

#In one embodiment, the large group of the conjugate is a protein. The protein is any readily available protein, which is preferably inexpensive and which contains large numbers of lysine for hapten conjugations. Examples of suitable proteins in the art include bovine serum albumin (BSA), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH). Proteins may include enzymes, secretory proteins, globular proteins. A preferred protein for use herein is ovalbumin (OVA). Where the protein is an enzyme, it is preferred that it be selected from the group comprising alkaline phosphatase, glucose oxidase, horseradish peroxidase and amine enriched horseradish peroxidase.