Affinity Particle And Affinity Separation Method

Abstract

The present invention is affinity particles that are characterized by having phosphorylcholine groups represented by the following formula (1) covalently bonded onto the surface of inorganic powder and also by having ligands having specific affinity with a certain target substance covalently bonded or adsorbed onto the surface of inorganic powder. The object of the present invention is to provide an affinity separation method that uses affinity particles utilizing inexpensive inorganic particles and is capable of separating the target substance easily and with high accuracy.

1 - 7 . (canceled) 
   
   
       8 . A method of affinity separation of a target substance using affinity particles comprising:
 (1) a first step whereby ligands are bonded onto the affinity particles, said affinity particles comprising:
 (a) inorganic particles; 
 (b) reactive groups or adsorptive groups bound onto the surface of the inorganic particles; 
 (c) phosphorylcholine groups represented by the following formula (1): 
   
     
       
         
         
             
             
         
       
       covalently bonded onto the surface of said inorganic particles; 
       (2) a second step whereby the affinity particles having the ligands bonded thereto prepared in the first step are either dispersed in a solution sample containing the target substance selectively, so as to capture the target substance by the ligands, or are disposed with a column as carriers, so as to provide an affinity column for recovering the target substance; and 
       (3) a third step whereby the captured target substance is recovered from the affinity particles. 
     
   
   
       9 . The method of affinity separation of a target substance using affinity particles of  claim 8 , wherein the inorganic particles comprise silica, titanium oxide, zinc flower, alumina, iron oxide, talc, mica, sericite, and gold colloid particles. 
   
   
       10 . The method of affinity separation of a target substance using affinity particles of  claim 8 , wherein said inorganic particles have an average particle size of 20 nm to 500 μm, and a specific gravity of 1.0 g/cm 3  or higher. 
   
   
       11 . The method of affinity separation of a target substance using affinity particles of  claim 8 , wherein, in the first step, the reactive groups or adsorptive groups comprise amino groups, said amino groups being primary or secondary amines. 
   
   
       12 . A method of manufacturing affinity particles capable of bonding with ligands, said method comprising:
 (a) introducing reactive and/or adsorptive groups onto the surface of inorganic particles; and   (b) covalently bonding phosphorylcholine groups represented by the following chemical formula (1):   
     
       
         
         
             
             
         
       
       via the reactive groups onto the surface of the inorganic particles. 
     
   
   
       13 . The method of manufacturing affinity particles capable of bonding with ligands of  claim 12 , wherein the reactive and/or adsorptive groups are selected from the group consisting of amino groups, hydroxyl groups, carboxyl groups, thiol groups, and aldehyde groups. 
   
   
       14 . The method of manufacturing affinity particles capable of bonding with ligands of  claim 12 , wherein the reactive and/or adsorptive groups onto the surface of inorganic particles are introduced onto the surface of the inorganic particles by:
 (a) introducing the inorganic particles into a solution of 3-aminopropyl trimethoxysilane; and   (b) heating the resultant solution for 5-7 hours at about 100° C., according to the following scheme:   
     
       
         
         
             
             
         
       
     
   
   
       15 . The method of manufacturing affinity particles capable of bonding with ligands of  claim 12 , comprising forming a chemical bond between silica particles and phosphorylcholine groups by dehydrating the hydroxyl groups on the surface of the silica particles and the Si—OMe portion of the compound of following formula (3) or formula (4): 
     
       
         
         
             
             
         
       
     
     wherein m=2-6. 
   
   
       16 . The method of manufacturing affinity particles capable of bonding with ligands of  claim 12 , wherein the the ligands are covalently bonded or adsorbed onto remaining amino groups not bound to phosphorycholine groups. 
   
   
       17 . The method of manufacturing affinity particles capable of bonding with ligands of  claim 12 , wherein said ligands are selected from the group consisting of antibodies, antigens, enzymes, substrates, receptors, lectin, peptides, DNA, RNA, aptamers, protein A, protein G, avidin, biotin, chelating compounds, and metal ions. 
   
   
       18 . A method of manufacturing affinity particles having ligands bound thereto, said method comprising:
 (a) introducing reactive and/or adsorptive groups onto the surface of inorganic particles;   (b) covalently bonding phosphorylcholine groups represented by the following chemical formula (1):   
     
       
         
         
             
             
         
       
       via the reactive groups onto the surface of the inorganic particles; and 
       (c) covalently bonding or adsorbing ligands having specific affinity with a target substance onto the surface of said inorganic particles. 
     
   
   
       19 . The method of manufacturing affinity particles having ligands bound thereto of  claim 18 , wherein the reactive and/or adsorptive groups are selected from the group consisting of amino groups, hydroxyl groups, carboxyl groups, thiol groups, and aldehyde groups. 
   
   
       20 . The method of manufacturing affinity particles having ligands bound thereto of  claim 18 , wherein the reactive and/or adsorptive groups onto the surface of inorganic particles are introduced onto the surface of the inorganic particles by:
 (a) introducing the inorganic particles into a solution of 3-aminopropyl trimethoxysilane; and   (b) heating the resultant solution for 5-7 hours at about 100° C., according to the following scheme:   
     
       
         
         
             
             
         
       
     
   
   
       21 . The method of manufacturing affinity particles having ligands bound thereto of  claim 18 , comprising forming a chemical bond between silica particles and phosphorylcholine groups by dehydrating the hydroxyl groups on the surface of the silica particles and the Si—OMe portion of the compound of following formula (3) or formula (4): 
     
       
         
         
             
             
         
       
     
     wherein m=2-6. 
   
   
       22 . The method of manufacturing affinity particles having ligands bound thereto of  claim 18 , wherein the ligands are covalently bonded or adsorbed onto remaining amino groups not bound to phosphorycholine groups. 
   
   
       23 . The method of manufacturing affinity particles having ligands bound thereto of  claim 18 , wherein said ligands are selected from the group consisting of antibodies, antigens, enzymes, substrates, receptors, lectin, peptides, DNA, RNA, aptamers, protein A, protein G, avidin, biotin, chelating compounds, and metal ions.