Chemiluminescence enhancer

Abstract

The present invention provides a chemiluminescence enhancer treated to retain favorable dispersibility of fine solid carriers and stably exert a chemiluminescence enhancing action. The invention provides a chemiluminescence enhancer used for signal detection in a solid phase immunoassay using antigen or/and antibody immobilized onto fine solid carriers dispersible in a liquid medium, consisting of a water soluble macromolecular quaternary ammonium salt, a quaternary sulfonium salt or a quaternary phosphonium salt in order to enhance emission of light caused by an enzymatic reaction of a chemiluminescent substrate having dioxetane, wherein the chemiluminescence enhancer is given an aggregation inhibition treatment of the fine solid carriers by the treatment with an oxidizing agent or a reducing agent, and a chemiluminescence method and a kit using the chemiluminescence enhancer.

1 . A chemiluminescence enhancer, the enhancer comprising at least one of a water soluble macromolecular quaternary ammonium salt, a quaternary sulfonium salt or a quaternary phosphonium salt capable of enhancing emission of light caused by enzymatic reaction of a chemiluminescent substrate having dioxetane each salt having been treated with an oxidizing agent or a reducing agent to provide aggregation inhibition of an antigen and/or an antibody immobilized on a solid fine carrier.  
     
     
         2 . The chemiluminescence enhancer according to  claim 1  wherein the enhancer does not substantially comprise a component with a molecular weight of more than 400,000 daltons in the molecular weights separated by an ultrafiltration method.  
     
     
         3 . The chemiluminescence enhancer according to  claim 1  wherein the chemiluminescent substrate is a substrate represented by the general formula:  
       
         
           
           
               
               
           
         
       
       wherein R 2  is an aryl group substituted with an X-oxy group, which forms a 1,2-dioxetane compound that is an unstable oxide intermediate when X is eliminated by an activator selected from acid, base, enzyme, organic or inorganic catalyst and electron donor to induce a reaction, which unstable 1,2-dioxetane compound is decomposed with releasing electron energy to produce light and two carbonyl-containing compounds of general formulae,  
       
         
           
           
               
               
           
         
       
       and X is a chemically easily reactive group that is eliminated by an enzyme; R 1  is selected from the group consisting of an alkyl group, an alkoxy group, an aryloxy group, a dialkylamino group, a trialkylsilyloxy group, an arylsilyloxy group, an aryl group and an aryl group that is bound to an aryl group R 2  to form a polycyclic aryl group with X-oxy group substitution, which spiro-binds to a 1,2-dioxetane ring; R 3  and R 4  are each an alkyl group or a heteroalkyl group, or R 3  and R 4  may be together bound to form a polycyclic alkylene group which spiro-binds to the 1,2-dioxetane ring.  
     
     
         4 . The chemiluminescence enhancer according to  claim 1  wherein the enhancer is prepared from a monomer selected from the group consisting of a quaternary ammonium salt, a quaternary sulfonium salt, a quaternary phosphonium salt and mixtures thereof.  
     
     
         5 . The chemiluminescence enhancer according to  claim 1  wherein the enhancer is a polymerized quaternary ammonium salt, a polymerized quaternary sulfonium salt, a polymerized quaternary phosphonium salt or copolymers thereof.  
     
     
         6 . The chemiluminescence enhancer according to  claim 1  wherein the enhancer is selected from the group consisting of poly[vinylbenzyl(benzylmethyl ammonium chloride)], poly(vinylbenzyltrimethyl ammonium chloride), poly[vinylbenzyl(tributyl ammonium chloride)], benzylmethylcetyl ammonium chloride, polymethacrylamidepropylenemethyl ammonium chloride, poly[vinylbenzyl(triethyl ammonium chloride)], poly[vinylbenzyl(2-benzyl-amino)ethyldimethyl ammonium chloride], poly[vinylbenzyldimethyl(2-hydroxy)-ethyl ammonium chloride], poly[vinylbenzyl(trimethylphosphonium chloride)], poly[vinylbenzyl-(tributylphosphonium chloride and poly[vinylbenzyl(trioctylphosphonium chloride)] and copolymers thereof.  
     
     
         7 . The chemiluminescence enhancer according to  claim 1  wherein the fine solid carrier is a particle.  
     
     
         8 . The chemiluminescence enhancer according to  claim 7  wherein the particle is a magnetic particle.  
     
     
         9 . The chemiluminescence enhancer according to  claim 1  wherein the reagent having an oxidation or reduction property is selected from the group consisting of ammonium persulfate, sodium sulfite, sodium hypochlorite, hydrogen peroxide, sodium metaperiodate, potassium permanganate and potassium dichromate.  
     
     
         10 - 15 . (canceled)  
     
     
         16 . In combination, a chemiluminescent substrate, an antigen and/or an antibody immobilized on a fine solid carrier, and a chemiluminescence enhancer having dioxetane, the enhancer comprising at least one of a water-soluble macromolecular quaternary ammonium salt, a quaternary sulfonium salt or a quaternary phosphonium salt capable of enhancing emission of light caused by enzymatic reaction of the chemiluminescent substrate having dioxetane, each salt having been treated with an oxidizing agent or a reducing agent to provide aggregation inhibition of the antigen and/or antibody immobilized on the fine solid carrier.