A method of quantitating free (unbound) human C5a complement protein (C5a) from a sample comprising: a. removing human C5 from the sample; wherein the sample is incubated with C5 specific biotinylated antibody coupled to magnetic beads and wherein the magnetic beads coupled to C5 specific biotinylated antibody bound to human C5 are captured with a magnet; b. binding biotinylated anti-C5a capture antibody to strepavidin-coated particles; wherein said biotinylated anti-C5a capture antibody is added by capillary action to a Gyros Bioaffy 200 CD comprising columns with the strepavidin-coated particles; wherein said CD is subjected to centrifugal force inside a Gyrolab xPlore or a Gyrolab XP instrument, thus driving the biotinylated anti-C5a capture antibody to the strepavidin-coated particles in the columns; c. capturing the free (unbound) C5a in the sample on the capture antibody; wherein the sample after step a. is added to the CD by capillary action; wherein said CD is subjected to centrifugal force inside the Gyrolab xPlore or a Gyrolab XP instrument, thus driving the sample after step a. to the biotinylated anti-C5a capture antibody bound to the strepavidin-coated particles in the columns; d. detecting the captured free C5a; wherein an AlexaFluor labeled anti-C5a detection antibody is added to the CD by capillary action, wherein said anti-C5a detection antibody binds C5a at a different epitope from the epitope bound by the capture antibody; wherein said CD is subjected to centrifugal force inside the Gyrolab xPlore or a Gyrolab XP instrument, thus driving the detection antibody to the free C5a bound to the capture antibody bound to the strepavidin-coated particles in the columns; and e. quantitating the captured free C5a using laser-induced fluorescence detection. a. removing human C5 from the sample; wherein the sample is incubated with C5 specific biotinylated antibody coupled to magnetic beads and wherein the magnetic beads coupled to C5 specific biotinylated antibody bound to human C5 are captured with a magnet; b. binding biotinylated anti-C5a capture antibody to strepavidin-coated particles; wherein said biotinylated anti-C5a capture antibody is added by capillary action to a Gyros Bioaffy 200 CD comprising columns with the strepavidin-coated particles; wherein said CD is subjected to centrifugal force inside a Gyrolab xPlore or a Gyrolab XP instrument, thus driving the biotinylated anti-C5a capture antibody to the strepavidin-coated particles in the columns; c. capturing the free (unbound) C5a in the sample on the capture antibody; wherein the sample after step a. is added to the CD by capillary action; wherein said CD is subjected to centrifugal force inside the Gyrolab xPlore or a Gyrolab XP instrument, thus driving the sample after step a. to the biotinylated anti-C5a capture antibody bound to the strepavidin-coated particles in the columns; d. detecting the captured free C5a; wherein an AlexaFluor labeled anti-C5a detection antibody is added to the CD by capillary action, wherein said anti-C5a detection antibody binds C5a at a different epitope from the epitope bound by the capture antibody; wherein said CD is subjected to centrifugal force inside the Gyrolab xPlore or a Gyrolab XP instrument, thus driving the detection antibody to the free C5a bound to the capture antibody bound to the strepavidin-coated particles in the columns; and e. quantitating the captured free C5a using laser-induced fluorescence detection. <EOS>