Method for producing complex of biotin-labeled cypridina (cypridina noctiluca) luciferase with streptoavidin and method for stabilizing the same

Abstract

The present invention relates to a method for producing Cypridina luciferase labeled with hydrophilic biotin, characterized in that a biotin reagent containing a polyalkylene glycol structure as a spacer is reacted with Cypridina luciferase, and biotin-labeled Cypridina luciferase wherein a sugar chain in Cypridina luciferase has been biotinylated.

1 . A method for producing  Cypridina  luciferase labeled with biotin, characterized in that a biotin reagent containing a polyalkylene glycol structure as a spacer is reacted with  Cypridina  luciferase. 
     
     
         2 . The method according to  claim 1  wherein polyalkylene glycol is polyethylene glycol. 
     
     
         3 . The method according to  claim 1  wherein a position corresponding to a lysine residue K180 and/or K203 in the  Cypridina  luciferase in SEQ ID NO:1 has been biotinylated. 
     
     
         4 . A method for producing  Cypridina  luciferase wherein a sugar chain moiety has been biotinylated, comprising a step of introducing an aldehyde group in the sugar chain moiety by treating  Cypridina  luciferase having a sugar chain with a periodate salt and a step of subsequently reacting the aldehyde group in  Cypridina  luciferase having the sugar chain with a biotinylation reagent which selectively reacts with the aldehyde group. 
     
     
         5 . Biotinylated  Cypridina  luciferase which is  Cypridina  luciferase labeled with biotin having either
 (i) a structure wherein  Cypridina  luciferase is linked with biotin through a spacer comprising a polyalkylene glycol moiety; or   (ii) a structure wherein  Cypridina  luciferase having a sugar chain is linked with biotin through the sugar chain.   
     
     
         6 . The luciferase according to  claim 5  wherein polyalkylene glycol is ethylene glycol. 
     
     
         7 . The luciferase according to  claim 5  wherein a position corresponding to a lysine residue K180 or K203 in the  Cypridina  luciferase in SEQ ID NO:1 has been biotinylated. 
     
     
         8 . The  Cypridina  luciferase labeled with biotin according to  claim 6  wherein the polyalkylene glycol structure is represented by the following formula:
   —(CH 2 CH 2 O) n—     
       wherein n represents an integer of 2 to 500. 
     
     
         9 . The  Cypridina  luciferase labeled with biotin according to  claim 8  wherein n is 2 to 100. 
     
     
         10 . A complex of the  Cypridina  luciferase labeled with biotin according to  claim 5  with a polyvalent avidin substance. 
     
     
         11 . The complex according to  claim 10  wherein the polyvalent avidin substance has been labeled. 
     
     
         12 . A stabilization composition of the complex according to  claim 10  comprising said complex and at least one stabilizer selected from the group consisting of surfactants and albumin. 
     
     
         13 . The composition according to  claim 12  which is in a form of a buffer solution. 
     
     
         14 . A method for stabilizing the complex according to  claim 10  characterized in that said complex and at least one stabilizer selected from the group consisting of surfactants and albumin are dissolved in a buffer solution. 
     
     
         15 . The method according to  claim 14  characterized in that the surfactant is combined at about 0.1 to 1%. 
     
     
         16 . A kit for immunoassays including a buffer solution comprising the complex according to  claim 10  and a stabilizer selected from the group consisting of surfactants and albumin. 
     
     
         17 . The kit according to  claim 16  which is for measurement by ELISA. 
     
     
         18 . A method for producing tagged  Cypridina  luciferase labeled with biotin, wherein a lysine-containing tag peptide is linked to a C terminus or N terminus of  Cypridina  luciferase and the lysine contained in the tag peptide is biotinylated using biotin protein ligase (EC. 6.3.4.15). 
     
     
         19 . A complex of the  Cypridina  luciferase labeled with biotin according to  claim 6  with a polyvalent avidin substance. 
     
     
         20 . A complex of the  Cypridina  luciferase labeled with biotin according to  claim 7  with a polyvalent avidin substance. 
     
     
         21 . A complex of the  Cypridina  luciferase labeled with biotin according to  claim 8  with a polyvalent avidin substance. 
     
     
         22 . A complex of the  Cypridina  luciferase labeled with biotin according to  claim 9  with a polyvalent avidin substance.