Apolipoprotein dilution

Published: November 14, 2022

Updated: February 12, 2024

Apolipoproteins are present at high levels in human and mouse serum and plasma. For ELISA quantification, such samples must therefore be diluted 5,000-200,000 times depending on the apolipoprotein analyzed.

General: Serum or plasma samples with EDTA are recommended. Other anti-coagulants are also possible, however, heparin containing samples will give higher apoE values due to displacement of proteoglycan bound apoE and citrate plasma may affect apoJ levels. Samples should be aliquoted and frozen. For longer storage, -80°C is recommended. Avoid repeated freezing-thawing cycles. Before dilution, it is recommended to remove visible precipitate by centrifugation. Precise pipetting is important! Please change pipet tips between the steps and ensure thorough mixing. We recommend samples to be assayed in duplicates, the indicated volumes are sufficient for duplicates. Sample dilutions can preferably be made in polypropylene tubes or plates. Prepare the dilutions as near as possible to the start of the assay.

For human sample use the Apo ELISA buffer: Due to the presence of heterophilic antibodies found in a majority of human individuals, it is important to dilute serum and plasma samples in Apo ELISA buffer. Heterophilic antibodies can cross-link the assay antibodies which results in a false positive signal. Mabtech’s Apo ELISA buffer prevents the heterophilic antibodies from cross-linking the capture and detection mAbs. The buffer is available as a 5x concentrated solution (product code: 3652-M2).

Triton X buffer necessary for apoB analysis: To prevent interference by different LDL-particle sizes, serum/plasma samples should be treated with Triton X-100. Dilute samples 2x with 1% Triton X followed by vortex for 5 seconds. Such treatment can be done on undiluted or up to 25x diluted serum/plasma samples. This treatment is not necessary for the apoB standard or for cell line produced samples. Triton X treatment of samples will not interfere with the analysis of other apolipoproteins.

Recommended dilutions: The recommended dilutions are based on serum samples from fasting healthy subjects. Samples containing levels of the analyte beyond the standard range will require other dilutions.

For mouse apoA1 use the Sample diluent: To enable a correct quantification of mouse apoA1, it is important to dilute serum/plasma samples and standard in the Sample diluent. The buffer is available as a 5x concentrated solution and is included in the ELISA Pro kits. The recommended dilutions of mouse serum/plasma samples are based on plasma samples from C57BL/6 and BALB/c. Samples containing levels of the analyte beyond the standard range will require other dilutions.

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