A modified ELISA assay differentiates CCL20 locked dimers from wild-type monomers

Abstract

A novel engineered CCL20 locked dimer (CCL20LD) is nearly identical to the naturally occurring chemokine CCL20 but blocks CCR6-mediated chemotaxis and offers a new approach to treat the diseases of psoriasis and psoriatic arthritis. Methods for quantifying CCL20LD serum levels are needed to assess pharmacokinetics parameters and evaluate drug delivery, metabolism, and toxicity. Existing ELISA kits fail to discriminate between CCL20LD and the natural chemokine, CCL20WT (the wild type monomer). Herein, we tested several available CCL20 monoclonal antibodies to be able to identify one clone that can be used both as a capture and a detection antibody (with biotin-labeling) to specifically detect CCL20LD with high specificity. After validation using recombinant proteins, the CCL20LD-selective ELISA was used to analyze blood samples from CCL20LD treated mice, demonstrating the utility of this novel assay for preclinical development of a biopharmaceutical lead compound for psoriatic disease.

Keywords: Cytokine; ELISA; Locked dimer; Psoriasis.

Fig. 1.

ELISA diagrams and modification for CCL20LD differentiation. A. Diagram (a) and an experimental example (b) for a traditional ELISA assay to detect cytokine human CCL20. B. (a) Diagram for how a modification of ELISA assay to detect CCL20LD but not CCL20WT. (b) Modified ELISA assay was performed for CCL20 LD and CCL20WT with concentrations ranging from 20 pg/ml to 62,500 pg/ml. The OD value versus concentration for both CCL20 WT and CCL20LD is shown in an interpolated standard curve generated by second order polynomial.

Fig. 2.

Assay accuracy and interference assessment for CCL20LD ELISA assay. A. (a) Comparison of OD values of CCL20LD in serum mixed at different ratios with standard diluent. Serial diluted CCL20LD in different matrix diluents, i.e., serum/diluent mixtures at the ratios of ½, 1/5, 1/10, or 1/20 were tested by CCL20LD ELISA to identify the Matrix interference. (b) Recovery of spiked CCL20LD in serum samples at different serum dilutions. Recovery rate is calculated for the percent of measured concentration in serum mixed samples to the expected concentration in diluent (percent values shown for the calculation with CCL20LD spiked at 5 ng/ml). Two dotted lines define the acceptable recovery rate between 80 and 120%. B. Probability evaluation of endogenous CCL20 interference from psoriatic specimen. (a) ELISA assay for CCL20LD in mouse serum spiked with and without CCL20 (50 pg/ml). OD values for serial diluted CCL20LD, ranging between 0 pg/ml and 12.5 ng/ml, showed no significant difference between the two groups (p = 0.1315). (b) CCL20LD ELISA assay was performed for plasma samples composed of both psoriasis patients and healthy controls (n = 3 in each group). OD values were shown for each sample spiked with, or without, CCL20LD protein.

Fig. 3.

Application of CCL20LD ELISA assay for mouse in vivo study. A. CCL20LD was injected via IV with the dose of 400μg per mouse. Serum is collected at 2 h after administration followed by CCL20LD ELISA assay. Serum concentration is shown for both CCL20LD injected mice and un-injected controls (n = 3). B. CCL20LD ELISA data for serum samples from mice treated with CCL20LD through different routes, i.e., intravenous (IV), subcutaneous (SC), and intraperitoneal (IP). C. CCL20LD Half-life determination by collecting mouse serum at multiple time points (0, 1, 8, 24 h) after SC delivery (n = 3 mice per time point). Blood was collected through cardiac puncture at time indicated.