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Use of the HitHunter EFC Chemiluminescent Detection Kits with LEADseeker Multimodality Imaging System

Key words: EFC chemiluminescence • HitHunter LEADseeker


Competitive pressure is forcing organizations to continually increase screening throughput. Yet accuracy and sensitivity remain vital concerns, particularly for common screens such as luminescence assays. LEADseeker™ Multimodality Imaging System was developed to meet these demands by combining excellent detection with the ability to image whole plates in less than one minute.

This application note describes the use of the HitHunter™ range of EFC chemiluminescent assays with the LEADseeker Multimodality Imaging System. The Caspase 3 and p38 MAPK binding assay kits were used as model systems. The data demonstrate that the HitHunter range of assay kits, which are based on enzyme fragment complementation technology, perform well in conjunction with the chemiluminescent readout of LEADseeker Multimodality Imaging System equipped with the supplied luminescence filter. Used together, the components support organizations interested in adding high and ultrahigh throughput chemiluminescent assays to their screening programs.

Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

HitHunter Caspase 3 EFC Chemiluminescent 90-0028-02
Detection Kit

HitHunter p38 MAPK Binding Assay EFC 90-0028-31(L)
Chemiluminescent Detection Kit


Other materials required
Luminescence 660 nm cut-off filter

384-well plates (non-treated, non-sterile, polystyrene, white)

DEVD-CHO inhibitor (BIOMOL)

SB203580 and SB239063 inhibitors (Calbiochem)


Method
1. Caspase 3 method
(Note: The assay was set up in triplicate and all reagents werehawed on ice. The assay was carried out at room temperature.)

1.1 Prepare all reagents as recommended by the kit manufacturer.

1.2 Prepare Caspase 3 enzyme standard curve working dilutions in assay buffer.

1.3 Prepare DEVD-CHO inhibitor curve working dilutions in assay buffer.

1.4 Create "No Protease" control wells by adding 15 µl assay buffer to appropriate wells.

1.5 Add 5 µl assay buffer to all enzyme standard curve wells.

1.6 Create Caspase 3 standard curve by adding 10 µl from the working dilutions (see 1.2 above) to appropriate wells (final concentration range = 0.4–100 pg/well)

1.7 Add 10 µl Caspase 3 enzyme from the appropriate working dilution (see 1.2 above) to all inhibitor curve wells (final concentration = 100 pg/well).

1.8 Create DEVD-CHO inhibitor curve by adding 5 µl from the working dilution (see 1.3 above) to appropriate wells (final concentration range = 0.1–100 000 nM).

1.9 Add 5 µl ED Reagent to all wells and incubate the assay 3 h at room temperature.

1.10 Add 15 µl EA Reagent and 15 µl CL Substrate to all wells and incubate the assay 1 h at room temperature.

1.11 Image the plates for 30 s on Leadseeker Multimodality Imaging System. (Note: Due to the excellent sensitivity of LEADseeker Multimodality Imaging System, a reduced imaging time of 30 s is recommended to achieve the same results as described in the pack leaflet.)

2. p38 MAPK method
(Note: The assay was set up in triplicate and all reagents were thawed on ice. The assay was carried out at room temperature.)

2.1 Prepare all reagents as recommended by the kit manufacturer.

2.2 Create "No inhibitor" control wells by adding 20 µ l assay buffer to appropriate wells.

2.3 Create inhibitor curve wells by adding 20 µl SB 203580 or SB239063 inhibitor diluted in assay buffer to appropriate wells (final concentration range = 0.045–2650 nM)

2.4 Add 10 µl p38/EA Reagent mix to all the wells and incubate the assay 30 min at room temperature.

2.5 Add 10 µl ED Reagent to all wells and incubate the assay 30 min at room temperature.

2.6 Add 10 µl CL Substrate to all wells and incubate the assay 1 h at room temperature.

2.7 Image the plate on LEADseeker Multimodality Imaging System for 30 s. (Note: Due to the excellent sensitivity of LEADseeker Multimodality Imaging System, a reduced imaging time of 30 s is recommended to achieve the same results as described in the pack leaflet.)


Results
Caspase 3
The standard curves generated in two separate assays (Fig 1) were entirely consistent and comparable with the pack leaflet data. Both assay curves also gave excellent r2 values (0.99 and 0.96 for assay 1 and 2 respectively).

The IC50 results generated (Fig 2) were also consistent with the pack leaflet data. The value of 1.8 nM obtained was within 95% confidence intervals of the pack leaflet value (1.54 nM).

Z` data was generated by using the top standard (10 ng per well) as the positive control and the no-protease control as the negative control. Z` results are shown in Figure 3. The calculated Z` value of 0.67 confirms that the Caspase 3 assay is a suitable format for use with the LEADseeker Multimodality Imaging System.

p38 MAPK binding assay
The IC50 results generated for this assay (Fig 4) were consistent and comparable with the pack leaflet data. The values of 32 nM and 37 nM for the inhibitors SB203580 and SB239063, respectiv ely, were within 95% confidence intervals of the pack leaflet values (29 nM and 33 nM, respectively).


Conclusion
HitHunter Caspase 3 and p38 chemiluminescent assays are fully compatible with LEADseeker Multimodality Imaging System.

Enzyme titration, inhibition assay, and Z` analyses all demonstrate that these assays perform to pack leaflet specifications on LEADseeker Multimodality Imaging System.

LEADseeker Multimodality Imaging System and HitHunter assay kits can be effectively used in combination by organizations seeking to run high and ultra-high throughput luminescence assays.




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