An Enzyme-linked Immunosorbent Assay for the Detection of Complement Components on Red Blood Cells

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Abstract
A new technic using the principle of enzyme-linked immunoassay (ELISA) has been developed for the detection of complement components on red blood cells sensitized in vivo or in vitro. Using a double-antibody technic, anticomplement antisera (anti-C3c or anti-C3c/C3d) produced in rabbits was incubated with the red blood cells, followed by incubation with antirabbit alkaline phosphatase conjugated antiglobulin. The amount of the enzyme fixed was measured spectrophotometrically by the enzymatic hydrolysis of the substrate PNPP. A calibration curve was made from red blood cells on which complement was deposited by the method of Fruitstone. The technic showed a greater sensitivity than the standard antiglobulin tests and allowed simultaneous qualitative and semiquantitative estimates. The technic can be performed in any laboratory equipped with the standard equipment found in a blood bank, including a spectrophotometer. The authors made a modification of Alsever’s solution, which allowed the safe and stable preservation of complement coated red blood cells for 15 days. Significant positive results were obtained clinically using this technic, while negative or weakly positive reactions were obtained by the conventional antiglobulin tests.