Establishment and Qualification of A Competitive Enzyme-Linked Immunosorbent Assay for the Measurement of Heparin in Plasma Products

Abstract

Abstract Heparin is used in plasma fractionation processes to prevent activation of coagulation factors. Consequently, intermediates and final products have to be measured for their heparin content. Established chromogenic heparin assays rely on measurement of the accelerated inhibitory action of antithrombin against thrombin or activated factor X. These assays, however, show limitations, especially at the low sample dilutions required to provide adequate sensitivity. These limitations, potentially biasing results, originate from the specific sample matrix including proteases or protease inhibitors or from the test sample’s color. To overcome these shortcomings, a commercially available competitive enzyme-linked immunosorbent assay (ELISA) was established, described so far to measure heparin in plasma only. Briefly, heparin contained in the sample competed with the binding of a peroxidase-labelled heparin binding protein to the wells of a heparin-coated plate. The six-point calibration curve ranging from 0.03 to 7.14 IU heparin/mL showed acceptable accuracy and reproducibility. Test samples were diluted at least 1/6 in normal human citrated plasma. Spike-recovery was carried out by adding heparin to obtain a concentration of 0.26 IU/mL in the test dilution of the respective sample. Acceptable recovery was found in all sample types investigated (final immunoglobulin G product and process intermediates, albumin and α1-proteinase inhibitor), while intra-and inter-run precision data showed relative standard deviations lower than 10%. The ELISA’s limit of quantification was 0.18 IU/mL, thus demonstrating required sensitivity. In summary, the competitive heparin ELISA allowed accurate, precise and sensitive heparin measurement in specific plasma protein matrices, which interfere with chromogenic heparin assays.