Determination of concentration of fk778 by competitive immunoassay

Abstract

Methods and kits for measurement of concentration of FK778 in a biological sample by means of an immunoassay, preferably a competitive immunoassay. In one aspect, the method and kit involve the use of (a) an antibody to FK778 conjugated to a label, e.g., an acridinium label, (b) an antibody to FK778 not conjugated to a label, (c) a solid phase containing an antibody to a first hapten, e.g., a fluorescein hapten, and (d) a bihapten comprising a first hapten and FK778 or an analogue of FK778, e.g., a bihapten comprising a fluorescein hapten and a FK778 hapten. In another aspect, the method and kit involve the use of (a) antibody to FK778, (b) a bihapten comprising FK778 or an analogue of FK778 and a first hapten, e.g., a bihapten comprising the fluorescein hapten and the hapten of FK778 or an analogue of FK778, and (c) a pretreatment reagent.

1 . A method for determining the amount of FK778 or metabolite of leflunomide in a test sample comprising the steps of: 
 (a) incubating a mixture comprising (1) a test sample suspected of containing FK778, (2) a diluent for the sample, if necessary, (3) a pretreatment reagent, (4) an antibody to FK778 or to an analogue of FK778, (5) a bihapten tracer comprising a first hapten and FK778 (or an analogue of FK778) to form a mixture comprising (i) unbound tracer, (ii) tracer bound to the antibody, (iii) FK778 bound to the antibody, and (iv) unbound FK778;    (b) measuring the ratio of tracer bound to the antibody and unbound tracer present by fluorescence polarization; and    (c) determining the amount of FK778 or analogue of FK778 in the test sample from the fluorescence polarization signal measured.    
   
   
       2 . The method of  claim 1 , wherein the bihapten comprises FK778 and a fluorescein derivative.  
   
   
       3 . A kit comprising (a) an antibody to FK778, (b) a bihapten tracer comprising a first hapten and FK778 or an analogue of FK778, and (c) a pretreatment reagent.  
   
   
       4 . The kit of  claim 3 , wherein the bihapten comprises FK778 and a fluorescein derivative.