The ability to assess whether an individual has been exposed to a pharmacological agent, and a capability of determining the concentration of such an agent in a biological sample is of broad importance in medicine, law enforcement and other areas. In particular, the narrow window of therapeutic utility, and the proximity of that window to toxicity for many drugs such as Procainamide have necessitated the development of assays capable of detecting and quantifying such substances.
Procainamide is a pharmaceutical agent having utility in treatment of irregular heartbeats. It functions to restore the heartbeat to a normal rhythm and to slow an overactive heart, thereby allowing the heart to work more efficiently. Procainamide produces its beneficial effects by slowing nerve impulses in the heart and reducing sensitivity of heart tissues. It is important to monitor procainamide levels to ensure that a patient is receiving the correct dose. This is particularly important with procainamide, as monitoring helps avoid such side effects of procainamide overdose as fast and irregular heartbeat, confusion, stupor, decreased blood pressure, fainting, and cardiac arrest. A reliable test for procainamide in the blood also is useful for assisting medical personnel in the diagnosis of the cause of less common procainamide-induced neurological side effects such as hallucinations, depression, and psychosis.
Immunoassays are assay systems that exploit the ability of an antibody to specifically recognize and bind to a particular target molecule, and operate by incubating an antibody that is capable of binding to a predetermined analyte molecule with a sample that is suspected of containing analyte. The concentration of the target molecule is proportional to the concentration of antibody-analyte immune complexes. In some immunoassays, the antibody is bound to a support. Free target molecule is allowed to react with the support, and the concentration of the target molecule is determined by measuring the concentration of antibody-analyte complex immobilized to the support.
Target molecules that have become bound to the immobilized antibody can be detected through the use of a labeled, second antibody that is capable of binding to a second binding site on the target molecule (i.e., a “sandwich” immunoassay). Immobilization of the labeled antibody on the support is proportional to the concentration of the target in the sample. Alternatively, in a competitive assay, the sample is incubated with a known amount of labeled target and an immobilized antibody. The target molecules in the sample compete with the labeled target molecules for the antibody binding sites. Thus, the concentration of antibody-bound labeled target molecules is inversely proportional to the concentration of target molecule in the sample.
The utility of an immunoassay in detecting an analyte depends upon its capacity to report the extent of the formation of immune complexes between the antibody employed and the analyte whose presence or concentration is being measured. In general, two independent approaches exist for increasing this capacity. The first approach involves labeling one or more of the reagents.
Another approach involves increasing the size of the immune complex to the point where it becomes capable of scattering light. In such cases, agglutination or turbidimetric immunoassay methods may be employed. Turbidimetric methods measure the reduction of light transmitted through the suspension of particles or aggregates. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. In turbidimetric assays, the rate of change in light scatter may also be measured, and provides an indication of the amount of antigen present.
Turbidimetric assays for therapeutic drugs and drugs of abuse which use hapten coated particles are commercially available. An example of such an assay is a particle-enhanced turbidimetric-inhibition immunoassay (PETINIA). This immunoassay format uses drug-hydrophilic linker-particle reporter reagents (particle reagents). These particle reagents utilize very small latex particles (e.g., 70 nm) to which have been attached a drug or other compound of interest. When viewed with monochromatic light having a wavelength (e.g., 340 nm) larger than the diameter of the suspended particles, e.g., the suspension is relatively transparent. Under optimal conditions, addition of antibody specific for the drug on the particles will cause the particles to agglutinate, forming insoluble complexes. These complexes cause the suspension to become turbid and scatter light. When an antibody is added to a sample containing drug/particle conjugates and free drug, free drug competes with particle-bound drug for antibody, thereby inhibiting both the rate and extent of agglutination. This provides the basis for quantifying the amount of drug in the sample. Specifically, in the present invention, procainamide analog-hydrophilic spacer-particle reagents and anti-procainamide antibodies enable a rapid, precise and accurate PETINIA method for determining the amount of procainamide present in biological fluids. Such methods, by enhancing both the rate of immune complex formation, and the size of the immune complex, provide more efficient and effective immunoassays for determining the concentration of medically important pharmacological agents, such as procainamide. The present invention provides reagents and methods for conducting such improved immunoassays.
It is an object of the invention to provide novel procainamide analogs having a linking group that may be used to immobilize them to a support, in particular polymeric support. It is another object of the invention to provide novel methods of synthesizing biologically active procainamide analogs from aminobenzoic acids. It is also an object of the invention to provide particulate procainamide analog/polymer conjugates that may be used to detect the presence of procainamide antibodies. It is a further object of the invention to provide an immunoassay for procainamide which functions by measuring changes in the turbidity of a solution or dispersion containing a known quantity of a particulate procainamide analog/polymer conjugate, a known quantity of a procainamide antibody, and a sample suspected to contain procainamide.
The foregoing objects and advantages of the invention are illustrative of those that can be achieved by the present invention and are not intended to be exhaustive or limiting of the possible advantages which can be realized. Thus, these and other objects and advantages of the invention will be apparent from the description herein or can be learned from practicing the invention, both as embodied herein or as modified in view of any variation which may be apparent to those skilled in the art. Accordingly, the present invention resides in the novel methods, arrangements, combinations and improvements herein shown and described.
USPTO Backgrounds
USPTO Backgrounds