Quantitation of analytes in whole blood

A method for quantitating the level of a preselected analyte present in a sample of blood of a human or animal patient, said sample comprising plasma and white blood cells, said method sequentially comprising:

• i) providing four aliquots of equal volume of said sample, indicated as aliquots A, B, C, and D;
• ii) providing aliquot B with an amount of anti-analyte antibody sufficient to form an immunocomplex with said analyte in the sample, to provide reaction aliquot B;
• iii) providing aliquot A as a control to reaction aliquot B without said anti-analyte antibody, to provide reaction aliquot A;
• iv) providing aliquot D with a maximal stimulatory amount of antigen, together with an amount of anti-antigen antibody of the sane class as that of the anti-analyte antibody sufficient to form an immunocomplex with said antibody, to provide reaction aliquot D;
• v) providing aliquot C, as a control to reaction aliquot D, with an amount of antigen equal to that provided in reaction aliquot D, but without said anti-antigen antibody, to provide reaction aliquot C;
• vi) incubating reaction aliquots A, B, C, and D for a time sufficient for any immunocomplexes formed in the reaction aliquots to react with the white blood cells and complement proteins in the plasma to produce oxidants;
• vii) contacting a chemiluminescent compound which reacts with said oxidants to generate light with reaction aliquots A, B, C, and D, prior to or after step vi);
• viii) measuring light emission from reaction aliquots A, B, C, and D over a predetermined time period;
• and
• ix) determining said level of said preselected analyte by use of a preestablished relationship which correlates response factor to analyte level, said response factor determined from said light emissions from said reaction aliquots A, B, C, D using the formula ∫(B-A)/∫( D-C) wherein said values A, B, C and D represent said light emission from said reaction aliquots, respectively.