Assay Solution AN0121

AN0121: Human IL-1a/IL-1 F1 ELISA with Optimiser™ Microplates

Assay Solution for R&D Systems Human IL-1a/IL-1 F1 DuoSet (Catalog DY200)

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 IL-1a/IL-1 F1 ELISA utilizing reagents from R&D Systems (Catalog Number DY200) has been successfully transferred from a conventional 96-well ELISA plate format to an Optimiser™ microplate platform to achieve the following key performance benefits.

• Sample Volume: 5 µl • Assay time: Total assay time ˜ 2 hours (˜ 4.5 hour savings) • Assay reagents: 80%-95% saving on antibody use 5-20 assays for the cost of 1 • Sensitivity/Range: 2x improvement (3.9 – 500 pg/ml) • Potential to increase sensitivity to ˜ 0.2 pg/ml • Potential to achieve > 3-log dynamic range: 3.9 – 5,700 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 IL-1a/IL-1 F1 reagents were tested in a high-binding NUNC plate according to the protocol provided by the vendor. The operating range was confirmed to be 7.81 – 500 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™-H 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 2 µg/ml of capture and 0.2 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* 2.0 100 19.2 95% (Run 20 Optimiser™ plates for the capture antibody cost of 1 conventional ELISA plate)** Optimiser™ 2.0 5 0.96 Detection antibody Conventional* 0.05 100 0.48 80% (Run 5 Optimiser™ plates for the detection antibody cost of 1 conventional ELISA plate)** Optimiser™ 0.2 5 0.096

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

 

Optimiser™-Based ELISA and Results for Human IL-1a/IL-1 F1:

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 (Hu) IL-1a capture antibody (2.0 µg/ml in OptiBind™-H); 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 IL-1a in OptiBlock™ and samples in matrix-specific diluent) Add 5 µl of OptiWash™; wait 10 min. Add 5 µl of biotinylated anti-Hu IL-1a detection antibody (0.2 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 IL-1a/IL-1 F1 concentration vs background-adjusted RFU using a 4-parameter curve fit.

The Optimiser™-based ELISA developed for Human IL-1a/IL-1 F1 and illustrated above utilizes an 8-point standard curve from 3.9 – 500 pg/mL. Within this range, the back-calculated concentrations of standards range from 81.6% of target concentration at 3.9 pg/mL to 98.3 to 104.5% of target for the remaining standards. Percent coefficients of variation ranged from 1.64 to 15.5%. (By comparison, the range for conventional ELISA utilizing the same reagents is 7.81 to 500 pg/mL (R&D Systems package insert for catalog DY200). The Optimiser™-based ELISA realized actual savings in time and labor (˜ 69%), capture antibody (˜ 95%) 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 ˜ 0.2 pg/ml using the 20x sample repeat loading method and/or the operating range can be extended to > 3 logs using the modified substrate ratio.

 

Materials Used:

 

Material	Vendor	Vendor’s Catalog #	Vendor Contact
Human IL-1a/IL-1 F1	R&D Systems	DY2001	1-800-343-7475
NUNC Maxisorp microplate	Thermo Scientific	456537	1-800-625-4327
Reagent Diluent	R&D Systems	DY995	1-800-343-7475
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, r-human IL-1a, 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.