Assay Solution AN0116

AN0116: Human G-CSF ELISA with Optimiser™ Microplates

Assay Solution for R&D Systems Human G-CSF DuoSet (Catalog Number DY214)

Victor Moore; Applied Science Group, MiCo BioMed, Cincinnati, OH USA

Introduction:

MiCo BioMed’ 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 (Hu) Granulocyte Colony Stimulating Factor (G-CSF) ELISA utilizing reagents from R&D Systems (Hu G-CSF DuoSet, Catalog Number DY214) has been successfully transferred from a conventional 96-well ELISA plate format to the Optimiser microplate platform to achieve the following key performance benefits.

• Sample Volume: 5 µl • Assay time: Total assay time ˜ 2 hours (˜ 4 hour savings) • Assay reagents: 80% – 90% saving on antibody use 5-10 assays for the cost of 1 • Sensitivity/Range: Equivalent sensitivity/range (31.3 – 2000 pg/ml) • Potential to increase sensitivity to ˜ 1.6 pg/ml • Potential to achieve > 2-log dynamic range: 31.3 ˜ 22,800 pg/ml

Optimiser™ Method Development Process:

Please refer to the Assay Transfer Guide (Technical Support section of MiCo BioMed’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 MiCo BioMed.

Step 1:	Run a conventional ELISA to verify that the assay reagents perform as stated by the vendor(s). The human G-CSF reagents were tested in a high-binding NUNC plate according to the protocol provided by the vendor. The operating range was confirmed to be 31.3 – 2000 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™-D allows for 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 4 µg/ml of capture and 0.8 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.

Antibody Method Ab Concentration (µg/mL) Ab Vol./well (µL) Ab/Plate (µg) Savings Capture antibody Conventional* 2 100 19.2 90% (Run 10 Optimiser™ plates for the capture antibody cost of 1 conventional ELISA plate)** Optimiser™ 4 5 1.92 Detection antibody Conventional* 0.2 100 1.92 80% (Run 5 Optimiser™ plates for the detection antibody cost of 1 conventional ELISA plate)** Optimiser™ 0.8 5 0.384

*Vendor recommendation **Using material provided in listed catalog #’s

Optimiser™-Based ELISA and Results for Human G-CSF:

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.

Brief assay protocol Add 5 µl of anti-human (G-CSF capture antibody (4 µg/ml in OptiBind™-D); incubate for 10 min at RT. Add 5 µl of OptiWash™; wait 10 min. Add 5 µl of OptiBlock™; incubate for 10 min at RT. Add 5 µl recombinant (r) standard, control, and samples; incubate 20 min at RT. (Prepare rHu G-CSF in OptiBlock™ and samples in matrix-specific diluent) Add 5 µl of OptiWash™; wait 10 min. Add 5 µl of biotinylated anti-Hu G-CSF detection antibody (0.8 µg/ml in OptiBlock™); incubate for 10 min at RT Add 5 µl of OptiWash™; wait 10 min. Add 5 µl of SAv-HRP (MiCo BioMed, diluted 1:150 in OptiBlock™); incubate for 10 min at RT Add 30 µl of OptiWash™; wait 10 min. Repeat step. Add 10 µl of OptiGlow™; wait 15 min; read.

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 G-CSF concentration vs background-adjusted RFU using a 4-parameter curve fit.

The standard curve range for the Optimiser™-based ELISA developed for human G-CSF is 31.3 – 2000 pg/mL. The range is equivalent to that of a conventional ELISA using the same reagents (R&D Systems’ TDS, Catalog Number DY214). The Optimiser™-based ELISA realized actual savings in time and labor (˜ 67%), capture antibody (˜ 90%) and detection antibody (˜ 80%) (Reagent Savings Table) when compared with a conventional ELISA using the same materials at the vendor’s recommended concentrations. A further ˜ 95% savings in sample volume would be expected. Using the methods outlined in Application Notes (Technical Support section of MiCo BioMed’s website), the sensitivity of this assay can be projected at ˜ 1.6 pg/ml using the 20x sample repeat loading method and/or the operating range can be extended to > 2 logs using the modified substrate ratio.

 

Materials Used:

 

Material	Vendor	Vendor’s Catalog #	Vendor Contact
Human G-CSF DuoSet	R&D Systems	DY2141	1-800-343-7475
NUNC Maxisorp microplate	Thermo Scientific	456537	1-800-625-4327
Optimiser™ microplate (with Holder) 2	MiCo BioMed	OPH-10	www.micobiomed-usa.com Product categories: • Optimiser™ microplates • OptiMax™ buffers
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, rHu G-CSF, 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.