Overview: How to perform the PyroGene rFC Assay

The PyroGene rFC Assay is a fluorogenic assay that requires a fluorescence microplate reader – such as the PyroWave® XM Fluorescence Reader, paired with the WinKQCL® Endotoxin Detection & Analysis Software – to measure endotoxin values. Prior to setting up the plate, a template is prepared in WinKQCL software. Figure 1 shows the WinKQCL software template that was developed for use with the ASSIST PLUS. This template allows up to 21 samples to be tested in duplicate on one plate, with paired PPCs. It is designed to provide optimal flexibility for varying numbers of samples, while still allowing use of an 8 channel pipette to deliver the rFC working solution to the plate, as described in the assay instructions for use.

Downloads: App note and protocols for automated set-up of the Lonza PyroGene® Recombinant Factor C (rFC) Assay for endotoxin detection on the ASSIST PLUS

A 5-300 μl D-ONE single channel pipetting module is first used to prepare standard dilutions. Following preparation of the standards, PPC is added to the designated wells using repeat dispense mode. Next, standards and samples are added to the plate in duplicate according to the above template, with samples added to both clean and PPC-spiked wells. Once all samples are added, the plate is pre-incubated at 37 °C for 10 minutes. During the incubation, the user prepares the working reagent in an INTEGRA 10 ml SureFlo™ reagent reservoir, which is added to the plate at the end of the pre-incubation period using a 300 μl VOYAGER 8 channel pipette on the ASSIST PLUS.

Tips:

• Use pyrogen-free certified GRIPTIPS® pipette tips in combination with SureFlo reservoirs to ensure accurate results. 10 ml SureFlo reservoirs require a dead volume of less than 30 μl.
• 300 μl long GRIPTIPS can access sample volumes of below 1 ml in 13x100 mm Lonza pyrogen-free test tubes, and will never leak or fall off.
• PPCs and samples can be dispensed using repeat dispense mode to save time and money.
• Plate layout of the blank, standards and samples in the WinKQCL software template is designed to offer the most flexibility for running full or partial plates.

Experimental set-up:

Deck Position A: LAL water – 10 ml multichannel reagent reservoir

Deck Position B: PyroGene rFC Assay plate – 96 well flat bottom plate (Corning)

Deck Position C: Standards, samples and PPC – INTEGRA tube rack

PyroGene rFC Assay plate set-up

Standards, samples and PPC-spiked samples are added to the plate

Pair the 0.5-300 μl D-ONE single channel pipetting module with the ASSIST PLUS pipetting robot. Place pyrogenfree test tubes containing 1 ml sample in the tube rack on deck Position C (blue tubes in Figure 2). Place three empty tubes in Positions A1-A3 within the rack. These are used to create the standard dilutions (magenta tubes in Figure 2). Place a tube holding 250 μl of 20 EU/ml endotoxin standard in A4 in the rack (green tube in Figure 2). Place a 10 ml SureFlo reservoir holding LAL water on deck Position A, and a 96 well flat bottom plate on deck Position B.

PyroGene rFC Assay reagent addition

Add 100 μl working reagent to each well of the plate

While the plate is pre-incubating, manually prepare the working reagent in a 10 ml SureFlo reservoir by combining fluorogenic substrate, rFC assay buffer and rFC enzyme solution in a 5:4:1 ratio. Place the working reagent in a clean 10 ml SureFlo reservoir on deck Position A (Figure 5). Pair a 300 μl 8 channel VOYAGER pipette with the ASSIST PLUS, and exchange the D-ONE tip deck for a standard tip deck. At the end of the 10 minute pre-incubation, place the plate on deck Position B. Initiate the VIALAB program 'Lonza PyroGene Assay reagent addition' to add 100 μl reagent to each well of the plate. When reagent addition is complete, place the plate in the fluorescence microplate reader to complete the assay.

Tips:

• VIALAB programs can be adapted to accommodate different numbers of samples, providing flexibility to meet current and future testing demands.
• For simplicity, 300 μl long GRIPTIPS are used in all steps of this assay.

Assay verification

Three runs of 21 samples plus standards were set up on the ASSIST PLUS, and tested according to the PyroGene rFC Assay instructions for use. Samples consisted of LAL water spiked with known concentrations of endotoxin standard. Five samples at each concentration were run on each plate, except for the 0 EU/ml sample, which was run 6 times per plate. Data analysis was performed using WinKQCL software. Table 1 shows the concentrations of standards and samples.

Table 1: Endotoxin concentrations of standards and spiked samples.

Results

The results are displayed in Tables 2 and 3. The standard curve for all runs displayed good linearity, and all curves were within the quality parameters as defined in the instructions for use (Table 2). Samples spiked with each concentration of endotoxin were detected (Figure 6). All unspiked samples remained undetectable at <0.005 EU/ml, which is the cut-off for acceptable endotoxin concentrations in pharmaceuticals and medical devices. All replicates of standards, samples and PPCs displayed a CV within the acceptable limit of less than 25 % (Table 3).

Table 2: Standard curve results with quality specifications for runs 1-3.

Table 3: Mean CV per plate for paired samples and PPCs. 

Remarks

• Partial plates: The supplied VIALAB programs can be adapted for partial plates, or for running samples in triplicate.
• Run report: VIALAB programs can be started directly from a PC connected to the ASSIST PLUS pipetting robot. A report is automatically generated after the run, documenting details such as start and end times, user identification, calculated volumes and any errors that occurred. This offers a convenient way to fulfill regulatory requirements.