Abstract
Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV2 repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV2 We generated hybridomas, screened them, and successfully produced a KIV2-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV9 without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence 4076LETPTVV4082 on KIV9 In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements.
Highlights
Supplementary key words lipoprotein (a) monoclonal antibody isoform kringle cardiovascular disease aortic stenosis metabolism, therapy lipoprotein (a)-kringle IV9
Plasma Lp(a) levels are genetically determined by the production and secretion rate of apo(a) by hepatocytes, with isoforms containing a small number of KIV2 repeats being secreted more efficiently, leading to an inverse association of KIV2 repeat number and plasma Lp(a) levels [2]
One colony was expanded in culture and the antibody LPA-KIV9 was purified and shown to be an IgG1 isotype
Summary
Apo(a) consists of 10 unique kringle (K)IV repeats that are present in one copy, except for KIV2, which is present in a variable number of identical copies at the protein level (1 to >40). It contains one copy of KV and an inactive protease-like domain. Only one isoform-independent antibody binding to KIV9 on apo(a) has been described and well-characterized, but its peptide epitope has not been reported This antibody has been used to develop an ELISA method, and the ELISA has been used to assign a target value to the WHO/IFCC reference material, and it is used as a comparison method to validate commercial assays.
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