Protocol
Development and optimization of cytokine ELISAs using commercial antibody pairs

https://doi.org/10.1016/S0022-1759(01)00419-7Get rights and content

Abstract

The measurement of cytokines in plasma and other fluids often requires the use of an enzyme-linked immunosorbant assay (ELISA). In the research environment, a valuable assay is one that yields reliable results in the shortest amount of time for the least cost. To achieve this goal, a protocol has been outlined to develop sandwich ELISAs for cytokines using commercial antibodies. These guidelines for ELISA development include selecting antibody concentrations, choosing an appropriate buffer, reducing plasma interference and evaluating the optimal length for incubation periods. In addition, the protocol for a rapid IL-6 ELISA is presented. This ELISA allows measurement of IL-6 in a reduced amount of time by raising the concentration of antibodies used and increasing the temperature for incubation. By following the guidelines presented, cost-effective, cytokine ELISAs can be developed that yield low background, detect a wide range of concentrations, and are suitable for use in the research setting.

Introduction

Cytokines have been recognized as extremely important mediators in sepsis and other inflammatory processes. In many research studies, thorough examination of complex inflammatory conditions requires accurate measurement of cytokine levels in blood, lavage or tissue culture fluids and organ homogenates. The quantification of proteins, such as cytokines, may be achieved by several methods; however, the enzyme-linked immunosorbant assay (ELISA) is often the assay of choice in many studies.

The ELISA is frequently used to detect cytokines and other proteins because it offers many advantages over other assays, including ease of performance, availability of components and use of non-radioactive reagents Salonen and Vaheri, 1981, Boscato and Stuart, 1988. However, several factors may affect the ability of an ELISA to detect cytokines. Factors such as the composition of the reagents used (i.e. buffer pH and protein content) and the matrix analyzed (i.e. presence of heterophilic antibodies, plasma interference or non-specific binding) must be addressed to optimize the usefulness of an ELISA (Feldkamp, 1987). Many of these issues are resolved before assay use when commercial kits are available; however, these kits may be cost-prohibitive for the large-scale testing of research samples, making ELISA development an important process in the research setting.

Although standard ELISAs have advantages over many assays, the dynamic role of cytokines, such as IL-6, in many disease states can make the measurements obtained from the ELISA obsolete by the time the assay has been completed. In many cases, it would be advantageous to obtain measurements more rapidly, allowing decision-making based on cytokine levels obtained shortly after sampling (O'Reilly et al., 1999). Since it is possible to accelerate antigen–antibody interaction by modifying temperature and concentration (Chan, 1987), these principles may be useful in the development of rapid ELISAs.

This protocol will demonstrate the steps involved in the development and optimization of standard sandwich ELISAs for cytokines and a rapid IL-6 ELISA employing commercial matched antibody pairs. The primary goal was to produce cost-effective, cytokine ELISAs with low background and capable of detecting a wide range of concentrations, suitable for use in the research setting.

  • 1.

    Measurement of cytokines in biological and tissue culture fluids.

  • 2.

    Rapid measurement of IL-6 with potential use to determine experimental outcome and the course of therapeutic interventions.

Section snippets

Basic ELISA protocol

  • 1.

    incubating coated plate: overnight

  • 2.

    washing plate and loading samples and standards: 15 min

  • 3.

    incubating standards: 60 min

  • 4.

    washing plate and loading biotinylated antibody: 15 min

  • 5.

    incubating biotinylated antibody: 60 min

  • 6.

    washing plate and loading streptavidin–HRP solution: 15 min

  • 7.

    incubating streptavidin–HRP: 30 min

  • 8.

    washing plate and loading TMB: 15 min

  • 9.

    color development: 20–30 min

  • 10.

    stop reaction with sulfuric acid, reading plate and analyzing data: 10 min

Total time: 240–250 min.

Sequence for ELISA optimization

  • 1.

    Criss-cross dilution analysis

Basic ELISA protocol

  • 96-well microtiter plates (Nunc Immunoplate, Neptune, NJ)

  • matched anti-cytokine antibody pair (R&D Systems, Minneapolis, MN)

    • monoclonal antibody for capture

    • polyclonal biotinylated antibody

  • cytokine standards-recombinant proteins (R&D Systems)

  • blocking solution:

    • 2% (w/v) bovine serum albumin (Sigma) in PBS, pH 7.4

  • dilution buffer:

    • PBS with 0.05% (v/v) Tween 20 (Surfact-™Amps, 10% Tween 20; Pierce, Rockford, IL) and 2% (v/v) fetal calf serum (Biocell, Carson, CA)

  • streptavidin-conjugated to horseradish

Basic ELISA protocol

(i) The wells of 96-well microtiter plates are coated with commercial, anticytokine, monoclonal antibody (50 μl/well) diluted in PBS, pH 7.4. The plates are tapped gently to insure even distribution of the antibody solution over the bottom of each well and then incubated overnight at 4°C.

(ii) The plates are washed (5 washes, 250 μl/well, 15 s/wash) using an automatic plate washer. The remaining steps are conducted at room temperature on an orbital shaker unless otherwise indicated. The

Optimizing antibody concentrations

The results of the criss-cross analysis should reveal optimal antibody concentrations for the coating and detection antibodies, demonstrating low background and a large range of optical densities for the increasing amounts of standard. The antibody concentrations yielding good results are often lower than those recommended by the manufacturer. The use of lower antibody concentrations can result in a significant reduction in cost when a large number of assays are performed.

Comparison of blocking agents

When the different

Basic ELISA protocol

  • (i) Coat wells of plate with anti-cytokine antibody (50 μl/well) and incubate overnight at 4°C

  • (ii) Wash plate, add blocking solution (150 μl/well) and incubate for 1 h at room temperature

  • (iii) Wash plate, load samples and standards (50 μl/well) and incubate for 1 h at room temperature

  • (iv) Wash plate, load biotinylated antibody (50 μl/well) and incubate for 1 h at room temperature

  • (v) Wash plate, load streptavidin–HRP solution (1:20,000, 50 μl/well) and incubate for 30 min and room temperature

Discussion

In this paper, a protocol has been presented for the development of sandwich ELISAs using matched antibody pairs for the detection of cytokines in biological fluids. This protocol also presents an organized, step-wise, approach to maximize the performance of each ELISA. Of these steps, the selection of an appropriate buffer for initial blocking of non-specific binding sites and for use in a dilution buffer is the most important. The selection of an appropriate buffer can combat many factors

References (9)

There are more references available in the full text version of this article.

Cited by (0)

View full text