Methods, reagents and test kits for determination of subpopulations of biological entities

A method for analyzing a population of cells with lipid-containing membranes to determine the presence or quantity of a subpopulation of said cells having at least one characteristic antigen, within said population, comprising:

• (i) coupling said cells of said population nonselectively to an extractable reporter substance, said extractable reporter substance having a lipophilic moiety, whereby said lipophilic moiety of said extractable reporter substance stably associates with said lipid-containing membranes of said cells;
• (ii) contacting said cell population with an antibody which binds specifically to said at least one characteristic antigen of said subpopulation of cells, under conditions causing binding of said antibody to said antigen, said antibody being affixed to magnetic particles;
• (iii) magnetically separating said magnetic particles bearing said subpopulation of cells from said population;
• (iv) extracting said reporter substance from said separated subpopulation of cells or from the population remaining after separation of said subpopulation;
• and
• (v) detecting the level of extracted reporter substance in said separated subpopulation of cells, or in the population remaining after separation of said subpopulation to provide an indication of the presence or quantity of the subpopulation of cells having said characteristic antigen within said population.

A method for analyzing a population of cells with lipid-containing membranes to determine the presence or quantity of a subpopulation of said cells having at least one characteristic antigen, within said population, comprising:

• (i) coupling said cells of said population non-selectively to an extractable reporter substance said extractable reporter substance having a lipophilic moiety, whereby said lipophilic moiety of said extractable reporter substance stably associates with said lipid-containing membranes of said cells;
• (ii) contacting said cell population with (a) an antibody which binds specifically to said at least one characteristic antigen of said subpopulation of cells and (b) a specific binding substance which binds specifically with said antibody, said specific binding substance being affixed to a solid phase;
• (iii) separating from said population the solid phase bearing said subpopulation of cells;
• (iv) extracting said extractable reporter substance from said separated subpopulation of cells or from the population remaining after separation of said subpopulation;
• and
• (v) detecting the level of extracted reporter substance in said separated subpopulation of cells, or in the population remaining after separation of said subpopulation to provide an indication of the presence or quantity of the subpopulation of cells having said characteristic antigen within said population.

A method for analyzing a subpopulation of cells, having at least one characteristic determinant, present within a population of cells with lipid-containing membranes, said subpopulation of cells including individual subsets of interest, each said subset having at least one characteristic determinant, to determine the proportion of at least one subset of said cell subpopulation, comprising:

• (i) non-selectively coupling substantially all of said cells of said population to a detectable reporter substance said detectable reporter substance having a lipophilic moiety, whereby said detectable reporter substance stably associates with said lipid-containing membranes of said cells;
• (ii) contacting a first sample of said cell population from step (i) with a firs reagent comprising at least one specific binding substance which binds specifically to said at least one characteristic determinant of said cell subpopulation, under conditions causing binding of said first reagent to said cells of said subpopulation, thereby forming first complexes in said first sample;
• (iii) separating said first complexes in said first sample from unbound cells in said first samples;
• (iv) detecting the occurrence of said reporter substance in said first complexes;
• (v) contacting a second sample of said cell population from step (i), of equivalent volume and cell concentration to said first sample, with a second reagent comprising a specific binding substance capable of selectively interacting with said at least one characteristic determinant of said at least one subset of interest in said subpopulation, under conditions causing binding of said second reagent to said at least one characteristic determinant, thereby forming second complexes in said second sample;
• (vi) separating said second complexes in said second sample from unbound cells in said second sample;
• (vii) detecting the occurrence of a reporter substance in said second complexes;
• and
• (viii) determining the proportion of said at least one subset of interest in said cell subpopulation by quantitating the amount of said reporter substance stably associated with said second complexes relative to the amount of said reporter substance stably associated with said first complexes.

A method for analyzing a population of cells with lipid-containing membranes to determine the presence or quantity of a subpopulation of said cells, having a characteristic determinant, within said population, comprising:

• (i) coupling said cells of said population non-selectively to an extractable reporter substance, said extractable reporter substance having a lipophilic moiety, whereby said lipophilic moiety of said extractable reporter substance stably associates with said lipid-containing membranes of said cells;
• (ii) contacting said cell population with a specific binding substance which binds specifically to said determinant under conditions causing binding of said specific binding substance to said determinant, said specific binding substance being affixed to a solid phase;
• (iii) separating said solid phase, bearing said subpopulation, from said population;
• (iv) extracting said reporter substance from said separated subpopulation of cells;
• and
• (v) detecting the level of extracted reporter substance to provide an indication of the presence or quantity of the subpopulation of cells having said characteristic determinant within said population.

A method for determining the presence or quantity of a subpopulation of biological entities selected from the group consisting of eucaryotic cells, procaryotic cells and viruses, and having at least one characteristic determinant, within a population of said entities in a test sample, comprising:

• (i) coupling a detectable reporter substance non-selectively to said population of biological entities;
• (ii) contacting said biological entities in said test sample with a specific binding substance which binds specifically to said at least one characteristic determinant of said subpopulation of biological entities, under conditions causing binding of said binding substance to said at least one characteristic determinant;
• (iii) separating said specific binding substance bearing said subpopulation of biological entities, from said population;
• and
• (iv) simultaneously detecting the occurrence of said detectable reporter substance in a plurality of said biological entities in said separated subpopulation of biological entities in said test sample or in a plurality of said biological entities in said population of said entities remaining in said test sample after separation of said subpopulation, thereby to provide an indication determinative of the presence or quantity of said subpopulation of biological entities having said at least one characteristic determinant within said population of biological entities having said at least one characteristic determinant within said population.