Ip-10 based immunological monitoring

Abstract

The present invention relates to an immunological method and, more particularly, a method for measuring cell-mediated immune reactivity (CMI) in mammals based on the production of IP-10. The invention further discloses an assay and a kit for measuring CMI to an antigen using whole blood or other suitable biological samples. The methods of the present invention are useful in therapeutic and diagnostic protocols for human, livestock and veterinary and wild life applications, thus the invention further relates to a method for diagnosing an infection in a mammal.

1 . A method for diagnosing an infection caused by Mycobacteria comprising:
 a) incubating a sample comprising T-cells obtained from a mammal with at least one Mycobacteria specific peptide or protein test-antigen;   b) determining a test-antigen specific cell-mediated immune response by measuring the IP-10 level in said sample;   c) comparing the determined IP-10 level with a reference-level; and   d) determining whether said mammal is likely to be infected with at least one Mycobacteria, if the determined IP-10 level is equal to or above the reference level and/or determining whether said mammal is unlikely to be infected if said determined IP-10 level is below the reference level, with the provisos that said test-antigen is not PPD and said mammal is not a human.   
     
     
         2 . The method according to  claim 1 , wherein the sample is divided into at least two fractions and further comprising:
 a) incubating the first fraction of the sample with at least one Mycobacteria specific peptide or protein test-antigen to generate a response sample;   b) incubating the second fraction of the sample with an inactive solution to generate a nil sample;   c) determining the IP-10 level in the two fractions;   d) determining a test-antigen dependent cell-mediated immune response of the sample by subtracting the IP-10 level determined in the nil sample from the IP-10 determined in the response sample;   e) comparing the test-antigen dependent cell-mediated immune response or a value derived therefrom with the reference-level or a value derived therefrom; and   f) determining whether said mammal is likely to have an infection if said determined test-antigen specific cell-mediated immune response level is equal to or above the reference level and/or determining whether said mammal is unlikely to have an infection if said determined test-antigen specific cell-mediated immune response level is below the reference level.   
     
     
         3 . The method according to  claim 2 , further comprising dividing the sample into 3 fractions and incubating the third fraction of the sample with a T-cell activator to generate a positive control. 
     
     
         4 . The method according to  claim 1 , wherein the infection is an active infection, a latent infection, a recent infection, and/or a long term latent infection. 
     
     
         5 . The method according to  claim 1 , wherein the Mycobacteria is selected from the group consisting of  M. tuberculosis  complex organisms, a Mycobacteria where the region of difference (RD1) has not been deleted, and a Mycobacteria pathogenic to humans. 
     
     
         6 . The method according to  claim 1 , wherein the Mycobacteria specific peptide or protein test-antigen is selected from the group consisting of RD-1 antigens, ESAT-6, CFP10, TB7.7, Ag 85, and HSP-65. 
     
     
         7 . The method according to  claim 1 , wherein the sample is derived from blood. 
     
     
         8 . The method according to  claim 1 , wherein the sample is whole blood. 
     
     
         9 . The method according to  claim 1 , further comprising:
 a) determining the level of MCP-1 in response to the test-antigen specific stimulation;   b) combining the determined level of IP-10 and MCP-1; and   c) comparing said combined level with a combined reference level.   
     
     
         10 . The method according to  claim 1 , further comprising:
 a) determining the level of INF-γ and, optionally, MCP-1 and/or IL-2 in response to the test-antigen specific stimulation;   b) combining the determined level of IP-10 and INF-γ and, optionally, MCP-1 and/or IL-2; and   c) comparing said combined level with a combined reference-level.   
     
     
         11 . The method according to  claim 1 , wherein the mammal is selected from the group consisting of primates, livestock animals, laboratory test animals, and companion animals. 
     
     
         12 . The method according to  claim 11 , wherein the livestock animal is selected from the group consisting of sheep, cattle, pigs, horses, donkeys, and goats. 
     
     
         13 . The method according to  claim 11 , wherein the laboratory test animals are selected from the group consisting of mice, rats, rabbits, guinea pigs, and hamsters. 
     
     
         14 . The method according to  claim 11 , wherein the companion animals are selected from the group consisting of dogs and cats.