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TTP Instrument Finds Niche with Ratiometric Fluorescence MAP Kinase Protein Assay

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As companies develop newer, faster, and more comprehensive instruments for conducting cell-based assays, it is becoming clear that no platform can do everything. But researchers are quickly finding that some instruments are almost perfectly suited for particular types of assays.

An example of this is TTP Labtech’s Acumen Explorer laser scanning cytometer. Last week, at CHI’s Cell-Based Assays for High-Throughput Screening conference, TTP scientist Paul Wylie discussed how his company’s instrument could greatly reduce the time and effort involved in an important cell-signaling related assay.

Several companies described methods for increasing cell-based assay throughput at the CHI conference, held May 17-18 in Philadelphia, but Wylie’s method for rapidly determining the activation of MAP kinase proteins was deemed the most significant, winning the poster competition.

“It’s more novel for the speed we were doing the assay at,” Wylie told Inside Bioassays. “This particular type of assay has been done many times before, but using different, slower methods.”

According to Wylie, the two MAP kinase proteins ERK and JNK are part of a crucial three-tier cascade of cell-signaling, and are implicated in multiple disease states, particularly in inflammation. The proteins become activated via phosphorylation, and researchers typically attempt to detect this phosphorylated state.

The most traditional way of conducting such an assay, Wylie said, is to lyse the cells and perform a series of long and involved biochemical analyses — overall a slow, laborious method, he said.

“And the other way of doing it … is using two different antibodies that recognize the phosphorylated form of the kinase,” Wylie said. “I did this a lot with microscopes, and it gives you a nice little image … but again, these [assays] tend to be relatively slow.”

The significance of the Acumen Explorer-based assay was that Wylie and his colleagues were able to conduct a similar assay in about five minutes for an entire 384-well plate.

The key to speeding up the assay is the design of the Acumen Explorer, which Wylie says falls almost in between a microscope and a flow cytometer, although it is plate-based. Most plate-based readers involve CCD cameras that focus on the well and capture images, while a software suite analyzes the results. Although such instruments can be very fast, the camera must take some time to refocus on each area before taking a picture.

Wylie said that the Acumen uses a single laser, which scans from underneath the plate, and the resulting fluorescence is captured by photmultiplier tubes, so there is no need to refocus. In addition, Wylie was able to simultaneously view two fluorescent signals through the use of simple filter sets. The scanner is capable of simultaneously discerning four colors.

“You can actually characterize populations of cells while conducting the assay,” Wylie noted.

A downside is that the instrument does not capture actual images, but Wylie said that they can be reconstructed using the instrument’s software package. He also admitted that this is a perfect example of an instrument being well-suited to a particular assay.

“I’ve always argued that it’s really not competing with [other instruments], but that it’s more of an adjunct.”

The Acumen Explorer is available with a single 488-nm laser — optimized for green fluorescent protein — and will soon be available with a single 405-nm laser for other popular assays such as ß-lactamase and blue fluorescent protein. The company announced the newer version on April 29, and although it’s not on the market yet, “it’s been tested at a couple of sites, and we know what the data is like and that it’s working,” Wylie said.

A basic 488-nm model, with 2 PMTs and a slower read-card costs about £104,000 (about $186,000), according to Wylie, while a top model with all 4 channels of fluorescence and a faster read time costs about £154,000 ($276,000).

“We have used four channels of fluorescence at the same time,” he added, “but it makes assay development very challenging.”

— BB

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