Methods For Detecting And Analyzing N-Glycolylneuraminic Acid (NEU5GC) In Biological Materials

Abstract

The present application is in the field of sialic acid chemistry, metabolism and antigenicity. More particularly, the present invention relates to the detection and analysis of the non-human sialic acid, N-glycolylneuraminic acid (Neu5Gc) in biological materials, such as food and clinical specimens. Such detection and analysis is facilitated by the use of Neu5Gc specific antibodies. The present invention also relates to the detection of antiNeu5Gc antibodies in clinical samples, as well as the production of anti-Neu5Gc specific antibodies.

1 . A method of determining % N-glycolylneuraminic acid (Neu5Gc) of a biological material comprising the steps of:
 (a) measuring the amount of N-acetylneuraminic acid (Neu5Ac) present in the material per given weight;   (b) measuring the amount of Neu5Gc present in the material per given weight; and   (c) calculating the % Neu5Gc of the sample, wherein % Neu5Gc is determined using the formula: % Neu5Gc=[(Neu5Gc)/(Neu5Ac+Neu5Gc)]×100.   
     
     
         2 . The method of  claim 1 , wherein the biological material is a food sample. 
     
     
         3 . The method of  claim 2 , wherein the food sample is a red meat or a dairy product. 
     
     
         4 . The method of  claim 2 , further comprising the step of calculating the Neu5Gc
 content per serving by multiplying the amount of Neu5Gc in step (b) by a food serving size of a given weight.   
     
     
         5 . The method of  claim 1 , wherein the biological material is a clinical sample. 
     
     
         6 . The method of  claim 5 , wherein the clinical sample is from an animal source. 
     
     
         7 . The method of  claim 6 , wherein the clinical sample is urine, tissue, blood or saliva. 
     
     
         8 . The method of  claim 5 , wherein the tissue is suspected of being diseased. 
     
     
         9 . The method of  claim 5  further comprising the step of repeating steps (a) to (c) at more than one time period. 
     
     
         10 . The method of  claim 9 , wherein the clinical sample is from a human or non-human animal subject, and wherein the subject undergoes a dietary change or therapy during the time period. 
     
     
         11 . A method of purifying sialic acid-specific antibodies, comprising the steps of:
 (a) preparing antibodies to sialic acids;   (b) contacting the antibodies from step (a) with a first solid phase to which a sialic acid having a side chain has been attached; and   (c) contacting the antibodies that bound to the first solid phase with a second solid phase to which the sialic acid without the side chain has been attached.   
     
     
         12 . The method of  claim 11 , wherein the sialic acid having a side chain is selected from the group consisting of: N-acetylneuraminic acid, Ketodeoxynonulosonate, N-glycolylneuraminic acid, N-propanoylneuraminic acid, N-butanoylneuraminic acid, N-pentanoylneuraminic acid, N-hexanoylneuraminic acid, N-heptanoylueuraminic acid, N-oxohexanoylneuraminic acid, N-levulinolylnueuraminic acid, N-homolevulinoylneuraminic acid, N-oxohexanoylneuraminic acid, N-oxoheptanoylneuraminic acid, and N-oxooctanoylneuraminic acid. 
     
     
         13 . The method of  claim 11 , wherein the side chain is removed using periodate. 
     
     
         14 . A method of detecting anti-Neu5Gc specific antibodies in a biological material
 comprising the steps of:
 (a) determining to amount of anti-Neu5Ac antibodies present in the material using Neu5Ac as an antigen target; 
 (b) determining the amount of anti-Neu5Gc antibodies present in the material using anti Neu5Gc as an antigen target; and 
 (c) subtracting the amount of anti-Neu5Ac antibodies from the amount of anti-Neu5G-c antibodies to determine the amount of anti-Neu5Gc specific antibodies. 
   
     
     
         15 . The method of  claim 14 , wherein step (a) and step (b) are performed in identical systems, except that in step (a), Neu5Ac is the antigen and in step (b), Neu5Gc is the antigen target. 
     
     
         16 . The method of  claim 15 , wherein the biological material is a clinical sample. 
     
     
         17 . The method of  claim 16 , wherein the clinical sample is from a human subject. 
     
     
         18 . The method of  claim 17 , wherein the clinical sample is blood. 
     
     
         19 . An antibody specific for binding N-glycolylneuraminic acid (Neu5Gc) that is not truncated at carbon 6. 
     
     
         20 . A method for purification of antibodies specific to sialic acid groups comprising:
 (a) exposing an antibody containing solution to a first immobilized target that has a very low density of the sialic acid under conditions to allow antibody binding;   (b) challenging the bound antibodies with a second immobilized phase comprising the first immobilized phase treated with mild sodium periodate; and collecting the antibodies that do not bind to the second immobilized phase.   
     
     
         21 . A method for the diagnosis of disease comprising:
 (a) collecting a serum or tissue sample from an individual;   (b) analyzing the sample for an elevated level Neu5Gc specific antibodies in serum; and   (c) correlating an increased level of antibody with the presence of disease.   
     
     
         22 . The antibody of  claim 19 , prepared by a method comprising:
 (a) contacting a preparation that comprises sialic acid-specific antibody with a first solid phase comprising sialic acid-containing antigen,
 (i) wherein the side chain at carbon 6 of said sialic acid of said first solid phase is not truncated; 
 (ii) wherein said contacting is under conditions to allow specific binding of said sialic acid-specific antibody to said sialic acid to produce a first bound antibody; and 
 (iii) wherein said contacting produces a first flow through solution; 
   (b) eluting said first bound antibody of step (a) from said first solid phase to produce a first eluted antibody;   (c) contacting said first eluted antibody of step (b) with a second solid phase comprising sialic acid, wherein the side chain at carbon 6 of said sialic acid of said second solid phase is truncated, and wherein said contacting produces a second flow through solution; and,   (d) collecting antibody that does not bind to said second solid phase of step (c).   
     
     
         23 . The antibody of  claim 22 , said method further comprising, after step (a),
 (A) contacting said first flow through solution of step (a)(iii) with said first solid phase under conditions for specific binding of said sialic acid-specific antibody to said sialic acid to produce a second bound antibody and a third flow through solution, and   (B) collecting said second bound antibody.   
     
     
         24 . The antibody of  claim 23 , said method further comprising repeating, one or more times, steps (A) and (B). 
     
     
         25 . The antibody of  claim 22 , said method further comprising a pre-clearing step prior to step (a), wherein said pre-clearing step comprises contacting said preparation that comprises a sialic acid-specific antibody with a third solid phase comprising an antigen that lacks sialic acid, and collecting flow-through for use in the contacting step of step (a). 
     
     
         26 . The antibody of  claim 25 , wherein said antigen that lacks sialic acid comprises asialofetuin. 
     
     
         27 . The antibody of  claim 19 , wherein said antibody binds Neu5Gc-LacNAc-PAA but not Neu5Ac-LacNAc-PAA. 
     
     
         28 . The antibody of  claim 19 , wherein said antibody binds to bovine or chimpanzee sera, but not to human sera. 
     
     
         29 . The antibody of  claim 19 , wherein said antibody recognizes Neu5Gc independent of whether it is in a glycolipid or a glycoprotein. 
     
     
         30 . The antibody of  claim 19 , wherein said antibody recognizes Neu5Gc independent of whether the glycosidic linkage is α2-3 or α2-6. 
     
     
         31 . A Neu5Gc detection reagent comprising the antibody of  claim 19 .