AN0104: Human c-Met (Total) ELISA with Optimiser™ Microplates

Assay Solution for Invitrogen Human c-Met (Total) Antibody Pair (Catalog CHO0285)

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 c-Met (Total) ELISA utilizing reagents from LIFE Technologies (Invitrogen, Catalog Number CHO0285) 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: 90% saving on antibody use
10 assays for the cost of 1
• Sensitivity/Range: Equivalent sensitivity (0.78 – 50 ng/ml)
Potential to increase sensitivity to ≈ 0.039 ng/ml
Potential to achieve > 2-log dynamic range: 0.78 ~ 580 ng/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 c-Met (Total) reagents were tested in a high-binding NUNC plate according to the protocol provided by the vendor. The operating range was confirmed to be 0.78 – 50 ng/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 best performance is obtained when both capture and detection antibodies are diluted 1:125. 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 the 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 90%
Run 10 Optimiser™ plates for the coating
antibody cost of 1 conventional ELISA plate)**
Optimiser™ 1:125 5 NA
Detection antibody Conventional* 1:250 100 NA 90%
Run 10 Optimiser™ plates for the detection
antibody cost of 1 conventional ELISA plate)**
Optimiser™ 1:125 5 NA
*Vendor recommendation **Using material provided in listed catalog #’s

 

Optimiser™-Based ELISA and Results for Human c-Met (Total):

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 c-Met (Total) capture antibody (diluted 1:125 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 standard, control, and samples; incubate 20 min at RT. (Prepare Hu c-Met (Total) in OptiBlock™ and samples in matrix-specific diluent)
Add 5 μl of OptiWash™; wait 10 min.
Add 5 µl of anti-Hu c-Met (Total) detection antibody (diluted 1:125 in OptiBlock™); incubate for 10 min at RT
Add 5 μl of OptiWash™; wait 10 min.
Add 5 µl of anti-rabbit IgG-HRP (diluted 1:875 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 c-Met (Total) concentration vs background-adjusted RFU using a 4-parameter curve fit.

  • The standard curve range for the Optimiser™-based ELISA developed for human c-Met (Total) is 0.78 – 50 ng/mL. The range is equivalent to that of a conventional ELISA in which the same reagents were used (Invitrogen TDS, Catalog Number CHO0285).
  • The Optimiser™-based ELISA realized actual savings in time and labor (≈ 64%), capture antibody (≈ 90%) and detection antibody (≈ 90%) (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 ≈ 0.039 ng/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 c-Met (Total) Antibody Pair Invitrogen CHO02851 www.invitrogen.com
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, Hu c-Met (Total), and Anti-Rabbit IgG-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.