Immunochromatography detection of multidrug-resistant staphylococcus and diagnostic kit

Abstract

It is intended to provide an immunochromatographic detection device which detects PBP2′ specifically produced by a multidrug-resistant Staphylococcus bacterium by an immunochromatographic detection method sensitively, simply and rapidly and is capable of determining infection with a multidrug-resistant Staphylococcus , a diagnostic method using the detection device, and a diagnostic kit including the detection device. The invention is directed to the immunochromatographic detection device for detecting a multidrug-resistant Staphylococcus which includes a sample supply unit which supplies a sample solution which is assumed to contain a multidrug-resistant Staphylococcus or a solution which is assumed to contain PBP2′ released from a cell wall by a pretreatment of a sample onto a sheet-shaped solid-phase support, a labeling reagent unit which retains a labeling reagent in which an antibody specifically binding to PBP2′ is labeled spreadable on the solid-phase support, and a trapping reagent unit in which a trapping reagent capable of trapping a complex of PBP2′ and the labeling reagent by specifically binding to the complex is immobilized, and in which an amphoteric surfactant; an anionic surfactant and/or a nonionic surfactant is contained in the trapping reagent unit or in the solid-phase support upstream of the trapping reagent unit.

1 . A method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, comprising detection of a cell-wall-synthesizing enzyme, PBP2′, via immunochromatography detection based on an antigen-antibody reaction. 
   
   
       2 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 1 , which involves the use of an immunochromatography detection device comprising on a sheet-like solid-phase support: a sample supply site to which a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment is supplied; a labeled reagent site that holds a reagent, which is a labeled antibody binding specifically to PBP2′, in a manner such that the reagent is able to spread across the solid-phase support; and a capture reagent site on which a capture reagent capable of specifically binding to and capturing a complex of PBP2′ and the labeled reagent has been immobilized. 
   
   
       3 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 1 , wherein PBP2′ comes into contact with the labeled reagent at a site separated from a solid-phase support in advance, and a sample-reagent mixture comprising a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment and the labeled reagent that is a labeled antibody binding specifically to PBP2′ is supplied to the sample supply site. 
   
   
       4 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 2 , wherein the labeled reagent is an insoluble carrier to which an antibody is bound. 
   
   
       5 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 2 , wherein the capture reagent site comprises an ampholytic surfactant, an anionic surfactant, and/or a nonionic surfactant. 
   
   
       6 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 2 , wherein the capture reagent site comprises a sulfobetaine-type surfactant. 
   
   
       7 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 5 , wherein an anion having a high ionization tendency is added to a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment prior to supply of such solution to a sample supply site. 
   
   
       8 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 7 , wherein the anion having a high ionization tendency is at least one anion selected from the group consisting of a chloride ion, a bromide ion, and an iodide ion. 
   
   
       9 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 5 , wherein a cation having a high ionization tendency is added to a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment prior to supply of such solution to a sample supply site. 
   
   
       10 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 9 , wherein the cation having a high ionization tendency is at least one cation selected from the group consisting of a potassium ion, a calcium ion, a sodium ion, and a magnesium ion. 
   
   
       11 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 1 , which comprises a step of pretreating a sample via alkaline treatment or neutralization. 
   
   
       12 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 11 , wherein the alkaline treatment is carried out using an aqueous solution of alkali metal hydroxide or carbonate or an aqueous solution of alkaline earth metal hydroxide or carbonate. 
   
   
       13 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 12 , wherein the pH of the aqueous solution of alkali metal hydroxide or carbonate or the aqueous solution of alkaline earth metal hydroxide or carbonate is 11 or higher. 
   
   
       14 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 12 , wherein the concentration of the alkali metal hydroxide or carbonate or alkaline earth metal hydroxide or carbonate is between 0.01 N and 1.0N. 
   
   
       15 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 11 , wherein the aqueous solution after alkali treatment is neutralized with a buffer. 
   
   
       16 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 1 , wherein the sample supply site comprises glass fibers. 
   
   
       17 . The method for detecting a bacterium that produces a cell-wall-synthesizing enzyme, PBP2′, according to  claim 1 , wherein the bacterium producing a cell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistant  staphylococcus.    
   
   
       18 . An immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, comprising on a sheet-like solid-phase support: a sample supply site to which a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′ or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment is supplied; a labeled reagent site that holds a reagent, which is a labeled antibody binding specifically to PBP2′, in a manner such that the reagent is able to spread across the solid-phase support; and a capture reagent site on which a capture reagent capable of specifically binding to and capturing a complex of PBP2′ and the labeled reagent has been immobilized, the capture reagent site comprising an ampholytic surfactant, an anionic surfactant, and/or a nonionic surfactant. 
   
   
       19 . The immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to  claim 18 , wherein the capture reagent site comprises a sulfobetaine-type surfactant. 
   
   
       20 . The immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to  claim 18 , wherein the sample supply site comprises glass fibers. 
   
   
       21 . The immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to  claim 18 , wherein the bacterium producing a cell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistant  staphylococcus.    
   
   
       22 . A kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, comprising: the immunochromatography detection device for detecting a bacterium producing a cell wall synthesizing enzyme, PBP2′, according to  claim 18 ; and an anion or cation solution having a high ionization tendency to be added to a sample solution deduced to contain a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, or a solution deduced to contain PBP2′ released from the cell wall via sample pretreatment. 
   
   
       23 . The kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, according to  claim 22 , wherein the anion having a high ionization tendency is at least one anion selected from the group consisting of a chloride ion, a bromide ion, and an iodide ion. 
   
   
       24 . The kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, according to  claim 22 , wherein the cation having a high ionization tendency is at least one cation selected from the group consisting of a potassium ion, a calcium ion, a sodium ion, and a magnesium ion. 
   
   
       25 . The kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, according to  claim 22 , which further comprises an alkali solution for alkali treatment of a sample with alkali and a buffer for neutralization. 
   
   
       26 . The kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, according to  claim 25 , wherein the alkali solution is an aqueous solution of alkali metal hydroxide or carbonate or an aqueous solution of alkaline earth metal hydroxide or carbonate. 
   
   
       27 . The kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, according to  claim 26 , wherein the pH of the aqueous solution of alkali metal hydroxide or carbonate or the aqueous solution of alkaline earth metal hydroxide or carbonate is 11 or higher. 
   
   
       28 . The kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, according to  claim 26 , wherein the concentration of the alkali metal hydroxide or carbonate or alkaline earth metal hydroxide or carbonate is between 0.01N and 1.0N. 
   
   
       29 . The kit for detecting a bacterium producing a cell-wall-synthesizing enzyme, PBP2′, according to  claim 22 , wherein the bacterium producing a cell-wall-synthesizing enzyme, PBP2′, is a multidrug-resistant  staphylococcus.