Immunoassay for Detecting Zika Virus Infection

Abstract

The present invention relates to a method for detecting Zika vims (ZIKV) infection in a biological sample from a subject. The method comprises testing the sample for IgM- and IgG-ZIKV NSI antibodies and determining the ZIKV IgM and ZIKV IgG signal intensities; and scoring the sample as positive or negative for ZIKV infection based on the combined results of such determinations. The biological sample is preferably blood, serum, plasma, cerebrospinal fluid, saliva or urine.

What is claimed is: 
     
         1 . An in vitro method for the detection of Zika virus (ZIKV) infection in a biological sample from a subject, comprising the steps of:
 a) testing the sample for IgM antibodies specific to ZIKV NS 1 protein or an epitope or immunogenic fragment thereof and determining the ZIKV IgM signal intensity;   b) testing the sample for IgG antibodies to ZIKV NS1 protein or an epitope or immunogenic fragment thereof and determining the ZIKV IgG signal intensity;   c) establishing a first ZIKV IgM signal intensity threshold and a second ZIKV IgM signal intensity threshold, wherein the second ZIKV IgM signal intensity threshold is higher than the first ZIKV IgM signal intensity threshold;   d) establishing at least a third ZIKV IgG signal intensity threshold;   e) detecting the presence or absence of ZIKV infection in the sample according to the following criteria:
 i) a ZIKV IgM signal intensity lower than the first ZIKV IgM signal intensity threshold is indicative of the absence of ZIKA infection; 
 ii) a ZIKV IgM signal intensity higher than the second ZIKV IgM signal intensity threshold is indicative of the presence of ZIKA infection; 
 iii) when the ZIKV IgM signal intensity is higher than the first ZIKV IgM signal intensity threshold and lower than or equal to the second ZIKV IgM signal intensity threshold:
 iiia) a ZIKV IgG signal intensity lower than the at least third ZIKV IgG signal intensity threshold is indicative of the absence of a ZIKV infection; and 
 iiib) a ZIKV IgG signal intensity higher than the at least third ZIKV IgG signal intensity threshold is indicative of the presence of a ZIKV infection. 
 
   
     
     
         2 . The method according to  claim 1 , wherein step a) and/or step b) are repeated. 
     
     
         3 . The method according to  claim 1 , wherein step a) is carried out before step b). 
     
     
         4 . The method according to  claim 1 , wherein step a) is carried out after step b). 
     
     
         5 . The method according to  claim 1 , which also includes testing the sample for IgA antibodies specific to ZIKV NS1 protein or an epitope or immunogenic fragment thereof. 
     
     
         6 . The method according to  claim 1 , wherein one or both of said steps a) and b) comprises an immunoassay. 
     
     
         7 . The method according to  claim 1 , wherein the IgM antibodies to Zika virus NS1 protein or an epitope or immunogenic fragment thereof are captured on a solid surface by contacting the biological sample with IgM binding molecules coated on the solid surface, and are detected using the NS1 protein or epitope or immunogenic fragment thereof as the detection reagent. 
     
     
         8 . The method according to  claim 7 , wherein the IgM binding molecules are anti-human IgM antibodies. 
     
     
         9 . The method according to  claim 1 , wherein the IgG antibodies to Zika virus NS1 protein or an epitope or immunogenic fragment thereof are captured on a solid surface by contacting the biological sample with IgG binding molecules coated on the solid surface, and are detected using the NS1 protein or epitope or immunogenic fragment thereof as the detection reagent. 
     
     
         10 . The method according to  claim 9 , wherein the IgG binding molecules are anti-human IgG antibodies. 
     
     
         11 . The method according to  claim 7 , wherein the amount of IgM binding molecules coated on the solid surface which are contacted with the biological sample substantially corresponds to the amount of IgM antibodies which are present in the biological sample. 
     
     
         12 . The method according to  claim 7 , wherein the solid surfaces are magnetic beads. 
     
     
         13 . The method according to  claim 1 , wherein one or both of said steps a) and b) comprises a chemiluminescent immunoassay. 
     
     
         14 . The method according to  claim 13 , which employs an isoluminol derivative as a chemiluminescent label 
     
     
         15 . The method according to  claim 6 , wherein the immunoassay comprises:
 i) contacting the biological sample with IgM binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgM antibodies in said sample, thereby capturing the IgM antibodies in the sample,   ii) contacting the biological sample with IgG binding molecules coated on a solid surface in an amount corresponding substantially to the amount of IgG antibodies in said sample, thereby capturing the IgG antibodies in the sample, and   iii) detecting captured ZIKV IgM antibodies and ZIKV IgG antibodies specific to ZIKV NS1 protein employing a labeled ZIKV NS1 antigen, epitope or fragment thereof.   
     
     
         16 . The method according to  claim 15 , wherein the biological sample, before contacting the IgG binding molecules coated on the solid surface, is diluted by a dilution factor comprised between 10-fold and 500-fold. 
     
     
         17 . The method according to  claim 1 , wherein ZIKV IgM and/or ZIKV IgG signal intensities are determined by reference to a calibration curve. 
     
     
         18 . The method according to  claim 1 , wherein the biological fluid is blood, plasma, serum, urine, cerebrospinal fluid or saliva. 
     
     
         19 . The method according to  claim 1 , wherein the infection is a secondary flavivirus infection. 
     
     
         20 . The method according to  claim 1 , wherein the subject is a human. 
     
     
         21 . The method according to  claim 9 , wherein the amount of IgG binding molecules coated on the solid surface which are contacted with the biological sample substantially corresponds to the amount of IgG antibodies which are present in the biological sample. 
     
     
         22 . The method according to  claim 9 , wherein the solid surfaces are magnetic beads.