AN0126: Human IFN-gamma ELISA with Optimiser™ Microplates
Assay Solution for R&D Systems’ Human IFN-γ DuoSet (Catalog DY285)
Victor Moore; Applied Science Group, Siloam Biosciences, Inc., Cincinnati, OH USA
Siloam Biosciences’ Optimiser™-based ELISAs offer a rapid, sensitive, and specific chemifluorescent-based ELISA procedure for the measurement of analytes using very small sample volumes. The Optimiser™ plate is SBS/ANSI-compliant and is compatible with traditional 96-well microplate instrumentation.
A human IFN-γ ELISA utilizing reagents from R&D Systems (Catalog Number DY285) has been successfully transferred from a conventional 96-well ELISA plate format to an Optimiser™ microplate platform to achieve the following key performance benefits.
Optimiser™ Method Development Process:
Please refer to the Assay Transfer Guide (Technical Support section of Siloam’s website) for details on the method development process. Each process step listed below requires 1 microplate (conventional for Step 1) and represents 1 experiment. In Step 2, the optimal coating buffer is selected from a panel of 12 coating buffers developed by Siloam.
|Step 1:||Run a conventional ELISA to verify that the assay reagents perform as stated by the vendor(s). The Human IFN-γ reagents were tested in a high-binding NUNC plate according to the protocol provided by the vendor. The operating range was confirmed to be 15.62 - 1000 pg/ml as stated by vendor.|
|Step 2:||Select the optimal coating buffer for use with the Optimiser™-based ELISA. The coating buffer screening test showed that OptiBind™-E supports the most efficient binding of capture antibody to microfluidic channel surface.|
|Step 3:||Run antibody titrations to select the optimal capture and detection antibody concentrations. The titration study shows that 6 µg/ml of capture and 0.125 µg/ml of detection antibody exhibit best performance. As described further, this Optimiser™-based ELISA is more sensitive than a conventional plate ELISA and the user should determine the optimal antibody concentrations to maximize savings and achieve desired operating range.|
Note that while capture and detection antibody concentrations for Optimiser™-based ELISAs may be 2 -4 times greater than those used in conventional ELISAs; the total quantity of antibody used in Optimiser™-based methods is significantly lower than that used in conventional ELISAs due to the much smaller reagent volumes used in the Optimiser™-based methods. Furthermore, sensitivity can be readily “tuned” for Optimiser™-based ELISAs using the repeat loading approach and antibody use can be further minimized if desired.
Comparison of Antibody Requirements for Conventional and Optimiser™-based ELISAs.
Optimiser™-Based ELISA and Results for Human IFN-γ:
The detailed procedure for this Optimiser™-based ELISA is contained in a companion document (User Manual or Detailed Experimental Protocol). The materials used in the procedure are specified in the Materials Table.
The plate is read using an FLx800™ Fluorescence Microplate Reader (BioTek Instruments, Inc.) equipped with a 528/20 nm excitation filter and a 590/35 nm emission filter with a sensitivity setting of 44. The data is analyzed using Gen5™ software (BioTek Instruments, Inc.) and a standard curve is created by plotting Human IFN-γ concentration vs background-adjusted RFU using a 4-parameter curve fit.
|Material||Vendor||Vendor’s Catalog #||Vendor Contact|
|Human IFN-γ DuoSet||R&D Systems||DY2851||1-800-343-7475|
|NUNC Maxisorp microplate||Thermo Scientific||456537||1-800-625-4327|
|Reagent Diluent||R&D Systems||DY995||1-800-343-7475|
|Normal goat serum||Sigma Aldrich||G6767||1-800-325-3010|
|Optimiser™ microplate (with Holder) 2||Siloam Biosciences||OPH-10|
|Polypropylene v-bottom plate||OPT/FL-231|
|Coat buffer test panel 3||OMR-TEST|
|Buffer reagent pack (with substrate) 2||OMR-10-J|
|Streptavidin-HRP for Optimiser™||OMR-HRP|
|1 Contains capture and detection antibodies, r-human IFN-γ, and SAv-HRP.|
|2 Optimiser™ plates and corresponding OptiMax™ buffer reagents are also available in 2-plate and 50-plate configuration.|
| 3 OMR-TEST is required only for assay transfer.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.