Development and validation of an isoform-independent monoclonal antibody-based ELISA for measurement of lipoprotein(a).

The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV ₂ and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV ₉ . Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27-1,402 nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, ⁴⁰⁷⁶ LETPTVV ⁴⁰⁸² , on KIV ₉ . In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.
Competing Interests: Conflict of interest S. M. M., N. N., and S. A. are employees of Medpace Reference Laboratories. S. M. reports consulting roles for Roche, Denka, Novartis, and research. A. G., J. L. W., and S. T. are coinventors of monoclonal antibodies directed to Lp(a) owned by UCSD directed to Lp(a). J. L. W. and S. T. receive royalties from patents on oxidation-specific antibodies and of biomarkers related to oxidized lipoproteins and Lp(a) held by UCSD. S. T. and J. L. W. are cofounders and have an equity interest in Oxitope, Inc and its affiliates (“Oxitope”) as well as in Kleanthi Diagnostics, LLC (“Kleanthi”). The terms of this arrangement have been reviewed and approved by the University of California, San Diego, in accordance with its conflict-of-interest policies. S. T. has a dual appointment at UCSD and Ionis Pharmaceuticals. J. L. W. is a consultant to Ionis Pharmaceuticals and A. G. to Kleanthi Diagnostics. All other authors declare that they have no conflicts of interest with the contents of this article.
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