AN0123: Human TNF-α/TNFSF1A ELISA with Optimiser™ Microplates

Assay Solution for R&D Systems TNF-α/TNFSF1A DuoSet (Catalog DY210)

Victor Moore; Applied Science Group, Siloam Biosciences, Inc., Cincinnati, OH USA

Introduction:

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 TNF-α ELISA utilizing reagents from R&D Systems (Catalog Number DY210) 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: 95% saving on antibody use
20 assays for the cost of 1
• Sensitivity/Range: Equivalent performance (15.6 – 1000 pg/ml)
Potential to increase sensitivity to ˜ 0.8 pg/ml
Potential to achieve > 2-log dynamic range: 15.6 11,400 pg/ml

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 TNF-α reagents were tested in a high-binding NUNC plate according to the protocol provided by the vendor. The operating range was confirmed to be 15.6 – 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™-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 4 µg/ml of capture and 0.250 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* 4.0 100 38.4 95%
(Run 20 Optimiser™ plates for the capture
antibody cost of 1 conventional ELISA plate)**
Optimiser™ 4.0 5 1.92
Detection antibody Conventional* 0.250 100 2.4 95%
(Run 20 Optimiser™ plates for the detection
antibody cost of 1 conventional ELISA plate)**
Optimiser™ 0.250 5 0.12
*Vendor recommendation **Using material provided in listed catalog #’s

 

Optimiser™-Based ELISA and Results for Human TNF-α:

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) TNF-α capture antibody (4 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 TNF-α in OptiBlock™ and samples in matrix-specific diluent)
Add 5 µl of OptiWash™; wait 10 min.
Add 5 µl of biotinylated anti-Hu TNF-α detection antibody (0.25 µg/ml in OptiBlock™); incubate for 10 min at RT
Add 5 µl of OptiWash™; wait 10 min.
Add 5 µl of SAv-HRP (Siloam Biosciences; 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 TNF-α concentration vs background-adjusted RFU using a 4-parameter curve fit.

  • The Optimiser™-based ELISA developed for Human TNF-α utilizes an 7-point standard curve from 15.6 –
    1000 pg/mL. The back-calculated concentrations of standards range from 75.3% of target for the 15.6 pg/mL standard to 99.5 – 111.1% for the remaining standards. Percent coefficients of variation for the standards range from 0.646 to 14.5% (By comparison, the range for conventional ELISA utilizing the same reagents is 15.6 to 1000 pg/mL (R&D Systems package insert for catalog DY210).
  • The Optimiser™-based ELISA realized actual savings in time and labor (˜ 69%), capture antibody (˜ 95%) and detection antibody (˜ 95%) (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 Siloam’s website), the sensitivity of this assay can be projected at ˜ 0.8 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 TNF-α/TNFSF1A DuoSet R&D Systems DY2101 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 Siloam Biosciences OPH-10

www.siloambio.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 TNF-α, 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.