Elsevier

Biologicals

Volume 39, Issue 1, January 2011, Pages 29-32
Biologicals

Effect of storage conditions on the stability of recombinant human MCP-1/CCL2

https://doi.org/10.1016/j.biologicals.2010.09.003Get rights and content

Abstract

Monocyte chemoattractant protein-1 (MCP-1) is commercially available in a form of recombinant protein. This makes it more convenient to study the functions of MCP-1 and its involvement in many cell functions. However, when using MCP-1 in experimental studies, if the analysis is not performed immediately, the stability of recombinant MCP-1 may become an issue. In this study, the stability of recombinant MCP-1 at different concentrations and storage conditions was investigated. Results show that no significant loss of MCP-1 is observed when MCP-1 solutions were stored at non-freezing condition (4 °C) for seven days. However, for storage at freezing conditions (−20 °C or −81 °C), it appears that the first freeze-thaw cycle may contribute to some loss of MCP-1 in the solutions, and such loss may be concentration and time dependent. The effect of multiple freeze-thaw cycles for storage at freezing conditions was also examined. Data reveal that the second freeze-thaw cycle causes approximately 50% loss of MCP-1 in the solutions. This finding confirms that multiple freeze-thaw cycles should be avoided. The findings of this study provide an outline of how storage can affect the stability of recombinant proteins and should be taken into account during the evaluation of the concentration of recombinant proteins.

Introduction

Chemokines (chemotactic cytokines) are low molecular weight proteins (8–12 kDa) that play an important role in the migration of immune cells [1]. Their functions involve homeostatic mechanisms, e.g., lymphocyte trafficking or immune surveillance, as well as inflammatory activities, such as recruiting lymphocytes to an injured area [2], [3]. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) is a chemokine that is secreted by various cell types and is involved in the progression of many inflammatory-related diseases, including HIV, cancer, and atherosclerosis [4]. The key function of MCP-1 during inflammation is to mediate the recruitment, migration, and infiltration of monocytes to an infected site [5].

Since MCP-1 was first purified more than two decades ago [6], [7], [8], a number of studies have been done to characterize and relate it to the pathogenesis of many diseases [4], [5], [9]. With advancements in technology, MCP-1 is now commercially available for laboratory use in the form of recombinant protein. Recombinant MCP-1 is frequently used in many in vitro studies [10], [11], [12], [13], including those that focus on the transendothelial migration of monocytes [14], [15]. In our previous study, we used recombinant human MCP-1 to examine the formation of MCP-1 concentration gradients across the collagen matrix of a 3D in vitro vascular tissue model (Manuscript in review). Briefly, recombinant MCP-1 was added to the system, samples were collected from the system at various time points, and the concentration of MCP-1 in the samples were determined by ELISA. The gradients of MCP-1 in the collagen matrix were estimated based on the concentration data. To achieve an accurate estimation of the amount of MCP-1 in the system, it is important to measure accurately the concentration of MCP-1 in the samples collected from the system. Since the collected samples are stored at −81 °C until ready for analysis, this raises a concern about the loss of MCP-1 during storage.

Generally, it is suggested that recombinant MCP-1 should be stored at a high concentration (10 μg/ml or greater) to maintain the stability of the protein. During an experiment, recombinant MCP-1 is diluted to physiological relevant concentrations, with working concentrations in the pg/ml to ng/ml range [10], [11], [12], [13], [14], [15]. Consequently, samples that are collected from such experimental systems contain recombinant MCP-1 at lower concentrations than the suggested value for storage. Thus, if those samples are not analyzed immediately, some MCP-1 molecules in the samples may become unstable over time.

In the present study, we investigated the effect of storing conditions on the loss of MCP-1 in samples containing recombinant human MCP-1 at the working concentrations. The objectives of this study include determining 1) the loss of MCP-1 at different storage conditions and 2) the effect of multiple freeze-thaw cycles on the stability of MCP-1 in the samples. Results of this study were aimed to provide a guideline to handle samples that contain recombinant human MCP-1, but can also be applied to other recombinant proteins used at low working concentrations.

Section snippets

Materials

Recombinant human MCP-1 was purchased from R&D Systems (Minneapolis, MN). Dulbecco’s phosphate-buffered saline (D-PBS), Medium 199, and penicillin–streptomycin–glutamine (PSG) solutions were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Tween® 20 was purchased from Fisher Scientific (Pittsburgh, PA). Bovine serum albumin (BSA) was purchased from Sigma–Aldrich (St. Louis, MO). BD OptEIA™ Human MCP-1 ELISA Set was purchased from BD

Storage conditions

To determine the effect of storage conditions on the loss of MCP-1 in samples, MCP-1 solutions were prepared from recombinant human MCP-1 and complete medium, and stored at different conditions for seven days. ELISA was performed at the end of the storage time to determine the concentration of MCP-1. Results are represented in Fig. 1. As shown in the figure, there is no significant change in the concentration of MCP-1 among samples with the same initial concentration, except for the 50 ng/ml

Discussion

The use of recombinant MCP-1 in an in vitro study generally requires that the recombinant protein is diluted from the stock concentration in the μg/ml range to the working concentrations in the pg/ml to ng/ml range. For many studies, samples containing MCP-1 at the working concentrations are stored and analyzed at the end of an experiment to determine the concentration of MCP-1. The stability of recombinant MCP-1 in samples may not be an issue if the analysis can be performed immediately after

Acknowledgment

This work was supported by a grant from the National Institute of Biomedical Imaging and Bioengineering (1R15EB009527-01).

References (15)

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