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The ELISAONE assay performed on a multi-mode microplate reader from BMG LABTECH

Miriam Uppill, Antony Sheehan, Ron Osmond TGR BioSciences Pty Ltd 03/2011
  • ELISAONE™ is a new technology from TGR BioSciences to measure ELISA in fluorescence mode 
  • ELISAONE™ is both robust and sensitive as evidenced by the data presented for the detection of EGF, TNFa and IL-2.

Table of contents

Introduction

ELISAONE™ technology has been developed by TGR BioSciences, to provide a means of running high per-formance sandwich immunoassays in a user-friendly 96-well format. ELISAONE assays use a traditional immuno-sandwich format, but with a major difference. The analyte and both antibodies are added to the ELISAONE assay microplate at the same time, allowing solution-phase binding. After a short incubation period, unbound assay reagents and analytes are washed away, and only immunocomplexes containing both antibodies are detected. The whole process can take as little as 60 minutes to complete, and requires just a single wash step. In contrast to other ELISA formats, the target-specific antibodies needed for the assay are not pre-bound to the microplate itself. The binding of antibodies to the analyte takes place in solution, allowing for efficient binding. This not only reduces assay times, but also affords several other benefits to the user. As the antibodies are not fixed to the plate, assays for several different targets can be performed in different wells on the same microplate, side-by-side. Another important benefit is reagent lifetime – many ELISA kits need to be discarded one month after opening, largely because the antibody-coated plates are often not stable after opening. With ELISAONE however, the expensive assay reagents are in reusable bottles, and last a long time after the ELISAONE microplate has expired. The inexpensive microplate can simply be discarded and another ordered, as they’re all identical.


ELISAONE assays for EGF, TNFa and IL-2 developed by TGR BioSciences, were used here to demonstrate the robustness and high sensitivity of the ELISAONE technology.


Assay Principle

ELISAONE™ - Protocol Overview

Materials & Methods

  • Human EGF, IL-2 and TNFa were all from R&D  Systems
  • ELISAONE ™ microplates and assay reagents (Antibody Mix, Wash Solution and Substrate Mix) were from TGR BioSciences
  • Filter-based microplate reader was from BMG LABTECH

 

Method:

ELISAONE™ assays for EGF, TNFa and IL-2 developed by TGR BioSciences, were used here to demonstrate the robustness and high sensitivity of the ELISAONE  technology.


Briefly, analyte (50 μL) prepared in either BSA/PBS or RPMI containing 10% FBS depending on the  experiment, was dispensed into duplicate wells of an ELISAONE microplate. Antibody Mix (50 μL) – containing  2 antibodies specific for the target analyte – was added  to the wells immediately after the analyte. The wells were sealed with a plate seal, and the ELISAONE  microplates were incubated for 1 hr at room temp, with gentle shaking. During this time the antibodies specifically bind to the analyte. Unbound components  were aspirated from the wells, and the wells were washed manually with 3 x 200 μL of 1X Wash Solution. Substrate Mix (50 μL) was added to the wells, the microplate was covered with foil, and incubated for  10 min at room temp, with gentle shaking. The plates  were uncovered, and the fluorescence signal (Ex 540 nm and Em 590 nm) was measured.


For recovery experiments, the amount of analyte in the well was calculated from a standard curve run on the same plate, prepared from 10-fold dilutions of analyte ranging from 1000 pg/mL to 0.1 pg/mL for EGF, and 10 ng/mL to 0.1 pg/mL for IL-2 and TNFa. The standard curve was fitted to a 4-parameter sigmoidal dose  response equation, and the fitted equations were used to estimate the amounts of analyte recovered in each experiment.

 

Results & Discussion

1. Assay reproducibility and sensitivity

Three assays were assessed for reproducibility over 3 separate experiments. Each day, either recombinant human EGF (Fig 2A), recombinant human IL-2 (Fig. 2B) or recombinant human TNFa (data not shown) were diluted to various concentrations in PBS containing 0.5% BSA in 96-well ELISAONE assay plates (n=4 wells/concentration point). ELISAONE was added to the wells, and the assays were incubated for one hour, washed, and incubated with ELISAONE Substrate Mix for 10 min. The plates were read immediately using a POLARstar Omega microplate reader. The limit of detection (LOD) was approximated using the signal obtained with buffer-only controls + 3x standard deviations.

 

  Day 1 Day 2 Day 3
LOD (EGF) 0.1 pg/mL 0.4 pg/mL 0.4 pg/mL
LOD (IL-2) 1 pg/mL 4 pg/mL 1 pg/mL
LOD (TNFa) 4 pg/mL 1 pg/mL 1 pg/mL

 

 

2. Analyte recovery from tissue-culture supernatants

The assays ability to recover known amounts of analyte from tissue culture media was investigated.

Each day, either recombinant human EGF (Fig. 3A), recombinant human IL-2 (data not shown) or recombinant human TNFa (Fig. 3B) were diluted to concentrations representing more than a 3-Log range (20 pg/mL, 100 pg/mL, 500 pg/mL or 2500 pg/mL) in RPMI containing 10% FBS in 96-well ELISAONE assay plates (n=2 wells/concentration point). ELISAONE antibody mix was added to the wells, and the assays were incubated for one  hour, washed, and incubated with ELISAONE Substrate Mix for 10 min. The plates were read immediately. The assay was able to accurately recover analytes over a range  of 3-logs difference in concentration.


The recovery was determined using an analyte-specific standard curve, diluted in PBS containing 10% FBS, and fitted to a non-linear 4-parameter sigmoidal curve. These calculations could be easily performed using  the MARS evaluation software that interfaces with all BMG LABTECH microplate readers.

 

Conclusion

 

This application note establishes ELISAONE™ (TGR BioSciences: www.tgrbio.com) as a robust and sensitive technology for the detection of biomarkers in biological samples. Its ease of implementation and substantially reduced preparation time, as compared to traditional ELISA assays, represents a substantial advance in ELISA technology. The filter-based microplate reader from BMG LABTECH provides an easy-to-use  instrument that will measure this fluorescence based assay quickly, accurately and robustly.

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