Determination of the presence of an antigen, particularly HLA-B27 antigen, at the surface of a cell, particularly lymphocytes, by cytotoxicity test combined with fluorescent staining

A method for typing a HLA determinant beared at the surface of lymphocytes in a whole anticoagulated blood test sample, which comprises the following steps;

• (a) placing said blood test sample in the presence of an antibody which specifically binds said HLA determinant;
• (b) allowing immune complexes to be formed between said HLA determinant and antibody;
• (c) placing said immune complexes in the presence of complement;
• (d) allowing the of said immune complexes constant region of the antibody to fix the complement this fixation leading to the disruption of said lymphocyte cell membrane;
• (e) placing said cells, the cell membrane of which is disrupted in the presence of a fluorescent dye reagent which specifically binds intra-cellular components;
• steps (a) to (e) being simultaneous;
• (f) lysing erythrocytes contained in said blood test sample;
• and
• (g) detecting the presence of dyed lymphocytes as an indication of the presence of said HLA determinant at the surface of their cell membrane.

A method for detecting the presence of antigens of interest at the surface of the cell membrane of a cell population contained in a test sample, which comprises the following steps:

• (a) distributing a substantially equal amount of cells in three test tubes C.sup.+, test and C.sup.- ;
• (b) adding to tubes C.sup.+ and test a suitable amount of a first antibody and of a second antibody, respectively, said first antibody specifically binding an epitope which is common to all cells of said cell population, and said second antibody specifically binding an epitope which is essentially unique to said antigen of interest;
• (c) allowing immune complexes to be formed between said epitopes and antibodies;
• (d) adding complement to tubes C.sup.+, test and C.sup.- ;
• (e) allowing the constant region of said antibodies to fix the complement in tubes C.sup.+ and test;
• allowing the cells of tube C.sup.- to fix the complement, wherein fixation by non-specific components is measured;
• the fixation of the complement leading to the destruction of the cell membrane of the cells bearing said epitopes or non-specific components;
• (f) adding to tubes C.sup.+, test and C.sup.- a fluorescent dye reagent which specifically binds intra cellular components;
• the steps of (a) to (f) being simultaneous;
• (g) detecting the number of dyed cells in tubes C.sup.+, test and C.sup.- to obtain values C.sup.+, test and C.sup.-, respectively;
• and,
• (h) calculating the percentage value of (test--C.sup.-)/C.sup.+ .times.100;
• a percentage value (test--C.sup.-)/C.sup.+ .times.100 superior to 10% being an indication of the presence of said antigen of interest in said test sample.

A method for typing a HLA determinant borne at the surface of lymphocytes in a whole anti-coagulated test sample, which comprises the following steps:

• (a) distributing a substantially equal amount of blood cells in three test tubes C.sup.+, test and C.sup.- ;
• (b) adding to tubes C.sup.+ and test a suitable amount of a first antibody and of a second antibody, respectively, said first antibody specifically binding a HLA determinant which is common to all lymphocytes, and said second antibody specifically binding a HLA determinant which is essentially unique to a type of lymphocytes;
• (c) allowing immune complexes to be formed between said HLA determinants and antibodies;
• (d) adding complement to tubes C.sup.+, test and C.sup.- ;
• (e) allowing the constant region of said antibodies to fix the complement in tubes C.sup.+ and test;
• allowing the blood cells of tube C.sup.- to fix the complement, wherein fixation by non-specific components is measured;
• the fixation of the complement leading to the destruction of the cell membrane of the blood cells bearing said HLA determinants or non-specific components;
• (f) adding to tubes C.sup.+, test and C.sup.- a fluorescent dye reagent which specifically binds intra-cellular components;
• the steps of (a) to (f) being simultaneous;
• (g) detecting the number of dyed lymphocytes and blood cells in tubes C.sup.+, test and C.sup.- to obtain values C.sup.+, test and C.sup.- respectively;
• and
• (h) calculating the percentage value of (test--C.sup.-)/C.sup.+ .times.100;
• a percent value (test--C.sup.-)/C.sup.+ .times.100 superior to 10% being an indication of the presence of said HLA determinant in said blood test sample.