Methods of Detecting Alloantibodies Using HLA and Non-HLA Antigens

Abstract

Described herein are materials and methods for detecting alloantibodies using both HLA and non-HLA antigens in a single assay.

1 . A composition comprising a first collection of solid-phase substrates each coated with a different purified human leukocytes antigen (HLA) to represent the HLA antigen population of a single cell line and a second collection of solid-phase substrates each coated with at a different non-HLA antigen listed in Table 1 or Table 1A. 
     
     
         2 . The composition of  claim 1  wherein the different purified HLA antigens are Class I HLA antigens or Class II HLA antigens. 
     
     
         3 . (canceled) 
     
     
         4 . The composition of  claim 1 , wherein the non-HLA antigen is a fusion protein comprising at least one domain, wherein the domain is a signal peptide, a modified cytoplasmic domain, purification tag or detection tag. 
     
     
         5 . The composition of  claim 4  wherein the domain is the B2 signal peptide, HLA cytoplasmic domain, EK Tag, V5 Tag or DPD Tag. 
     
     
         6 - 10 . (canceled) 
     
     
         11 . The composition of  claim 1 , wherein each solid phase substrates is detectably distinguishable the other solid-phase substrates within the composition. 
     
     
         12 . (canceled) 
     
     
         13 . A kit for determining the percentage of panel reactive antibodies in serum of a subject against HLA antigens comprising a first collection of solid-phase substrates wherein each solid-phase substrate is coated with different purified HLA antigens to represent the HLA antigen population of a single cell line such that said collection simulates the distribution of HLA antigens in a normal human population and a second collection of solid phase substrates wherein each substrate is coated with different purified non-HLA antigens listed in Table 1 or Table 1A. 
     
     
         14 . (canceled) 
     
     
         15 . The kit of  claim 13  wherein the HLA antigens are selected such that the HLA antigens presented on the solid phase substrate comprise Class I HLA antigens so as to simulate the distribution of Class I HLA antigens in a normal human population. 
     
     
         16 . (canceled) 
     
     
         17 . The kit of  claim 13  wherein the first collection comprises 54 different Class I HLA antigens. 
     
     
         18 . The kit of  claim 17  wherein the 54 different Class I HLA antigens are purified from 30 different cell lines. 
     
     
         19 . The kit of  claim 13  wherein the first collection comprises 22 different Class II HLA antigens. 
     
     
         20 . The kit of  claim 13  wherein the non-HLA antigen is a fusion protein comprising at least one domain, wherein the domain is a signal peptide, a modified cytoplasmic domain, purification tag or detection tag. 
     
     
         21 . The kit of  claim 20  wherein the domain is the B2 signal peptide, HLA cytoplasmic domain, EK Tag, V5 Tag or DPD Tag. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . The kit of  claim 13 , wherein at least one solid-phase substrate is detectably distinguishable from at least one other solid phase substrate. 
     
     
         25 - 29 . (canceled) 
     
     
         30 . The kit of  claim 22  wherein said first collection comprises at least one 3 μm microbead presenting Class I HLA antigens and at least one 5 μm microbead presenting Class II HLA antigens. 
     
     
         31 . A method for determining the percentage of panel reactive antibodies in serum of a subject against human leukocyte antigens (HLA) antigens, said method comprising:
 a. contacting a first collection of solid-phase substrates subtypes and a second collection of solid-phase substrate subtypes with serum from said subject for a sufficient time for anti-HLA antibodies in said serum to bind to said HLA-antigens to form a complex, wherein each substrate subtype in the first collection is coated with different purified HLA antigens to present HLA antigens derived from a cell population of a single cell, wherein each substrate subtype of the second collection is coated with different purified non-HLA antigens listed in Table 1 or Table 1A,   b. detecting the presence of the complex to determine the presence or absence of panel reactive antibodies, and   c. determining the percentage of panel reactive antibodies in the serum.   
     
     
         32 . The method of  claim 31  wherein the detecting step comprises detecting labeled ligand bound to the complex to determine the presence or absence of panel reactive antibodies. 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 31  wherein the detection step comprises detecting the presence of the complex using a solid phase immunoassay or a multiplexed bead immunoassay. 
     
     
         35 - 38 . (canceled) 
     
     
         39 . The method of  claim 31 , wherein the first collection of substrates is selected such that the HLA antigens presented thereon simulate distribution of Class I HLA antigens in a normal human population. 
     
     
         40 . The method of  claim 31 , wherein said first collection of substrates comprises 54 different Class I HLA antigens. 
     
     
         41 . The method of  claim 31 , wherein said first collection of substrates comprises 54 different Class I HLA antigens purified from 30 different cells. 
     
     
         42 - 43 . (canceled) 
     
     
         44 . The method of  claim 31 , wherein the non-HLA antigen is a fusion protein comprising at least one domain, wherein the domain is a signal peptide, a modified cytoplasmic domain, purification tag or detection tag. 
     
     
         45 . The composition of  claim 44 , wherein the domain is the B2 signal peptide, HLA cytoplasmic domain, EK Tag, V5 Tag or DPD Tag. 
     
     
         46 . The method of  claim 31 , wherein each solid-phase substrate is detectably distinguishable from the other solid phase substrates within a collection. 
     
     
         47 - 49 . (canceled) 
     
     
         50 . The method of claim  35 , wherein said microbeads comprise a mixture of 3 μm microbeads presenting Class I HLA antigens and 5 μm microbeads presenting Class II HLA antigens. 
     
     
         51 . The method of  claim 31 , wherein the subject is a transplant or transfusion recipient. 
     
     
         52 . The method of  claim 31 , wherein the serum sample is collected before the subject has received a transplant or transfusion. 
     
     
         53 . The method of  claim 31 , wherein the serum sample is collected after the subject has received a transplant or transfusion.