AN0120: AKT [pS473] ELISA with Optimiser™ Microplates

Assay Solution for Invitrogen AKT [pS473] Antibody Pair (Catalog CHO0115)

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.

An AKT [pS473] ELISA utilizing reagents from LIFE Technologies (Invitrogen, Catalog Number CHO0115) 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 (˜ 3.5 hour savings)
• Assay reagents: 80% saving on antibody use
5 assays for the cost of 1
• Sensitivity/Range: 4x improvement (0.39 – 100 Units/ml)
Potential to increase sensitivity to ˜ 0.02 U/ml
Potential to achieve > 2-log dynamic range: 0.39 - 285 U/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 AKT [pS473] reagents were tested in a high-binding NUNC plate according to Siloam’s standard protocol for conventional ELISAs. The assay confirmed the immunoreactivity of the antibodies and AKT [pS473] standard.
Step 2: Select the optimal coating buffer for use with the Optimiser™-based ELISA. The coating buffer screening test showed that OptiBind™-G 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 showed that capture antibody diluted 1:62.5 and detection antibody diluted 1:250 gave the 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 their 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* 1:250 100 NA 80%
(Run 5 Optimiser™ plates for the capture
antibody cost of 1 conventional ELISA plate)**
Optimiser™ 1:62.5 5 NA
Detection antibody Conventional* 1:1000 100 NA 80%
(Run 5 Optimiser™ plates for the detection
antibody cost of 1 conventional ELISA plate)**
Optimiser™ 1:250 5 NA
*Vendor recommendation **Using material provided in listed catalog #’s

 

Optimiser™-Based ELISA and Results for AKT [pS473]:

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 AKT coating antibody (diluted 1:62.5 in OptiBind™-G); 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 standard, control, and samples; incubate 20 min at RT. (Prepare AKT [pS473] standard in Reagent Diluent and samples in matrix-specific diluent)
Add 5 µl of OptiWash™; wait 10 min.
Add 5 µl of AKT [pS473] detection antibody (diluted 1:250 in Reagent Dliuent); incubate for 10 min at RT
Add 5 µl of OptiWash™; wait 10 min.
Add 5 µl of anti-rabbit IgG-HRP conjugate (diluted 1:600 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 AKT [pS473] concentration vs background-adjusted RFU using a 4-parameter curve fit.

  • The standard curve range for the Optimiser™-based ELISA developed for AKT [pS473] is 0.39 – 100 U/mL. The Optimiser™-based ELISA is 4-fold more sensitive than a conventional ELISA utilizing the same reagents (Invitrogen TDS for CHO0115).
  • The Optimiser™-based ELISA realized actual savings in time and labor (˜ 65%), capture antibody (˜ 80%) 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 Siloam’s website), the sensitivity of this assay can be projected at ˜ 0.02 U/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
AKT [pS473] Antibody Pair Invitrogen CHO01151 www.invitrogen.com
Reagent Diluent R&D Systems DY995 1-800-343-7475
NUNC Maxisorp microplate Thermo Scientific 456537 1-800-625-4327
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
1 Contains capture and detection antibodies, AKT [pS473] standard, and Anti-Rabbit lgG-HRP conjugate.
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.