Overview: How to perform the PyroGene® rFC endotoxin detection assay

The PyroGene® Recombinant Factor C (rFC) Endotoxin Detection Assay is a fluorogenic assay that requires a fluorescence microplate reader – the PyroWave® XM Reader paired with 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 samples plated by the ASSIST PLUS. This template allows up to 21 samples to be tested in duplicate on one plate with paired PPC samples. It is designed to provide the most flexibility for varying numbers of samples, while still allowing use of an 8 channel multichannel pipette to deliver rFC working solution to the plate, as written in the instructions for use.

Downloads: App note and protocols for automated set-up of the Lonza PyroGene® Recombinant Factor C Assay Endotoxin Detection Test on the ASSIST PLUS

The D-ONE 5-300 µl single channel pipetting module is first used to prepare standard dilutions. Following preparation of the standards, PPC is added to the designated wells of the 96 well plate in a repeat dispense mode. Next, standards and samples are added to the plate according to the above template. Standards and samples are added to the plate in duplicate, 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. At the end of the 10 minute pre-incubation, the working reagent is added to the plate using a VOYAGER 8 channel 300 µl pipette on the ASSIST PLUS.

Tips:

• Use pyrogen-free certified GRIPTIPS® in combination with SureFlo reservoirs to ensure accurate results. 10 ml SureFlo reservoirs require a dead volume of less than 30 µl.
• LONG 300 µl GRIPTIPS can access sample volumes of below 1 ml in 13 x 100 mm Lonza pyrogen‑free test tubes, and will never leak or fall off.
• PPC and samples are 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 spike – INTEGRA tube rack

1. PyroGene® rFC assay plate set-up

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

Pair the D-ONE 0.5-300 µl single channel pipetting module with the ASSIST PLUS pipetting robot. Place pyrogen-free 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 fluorescence plate on deck position B.

2. PyroGene® 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 the 300 µl 8 channel VOYAGER pipette with the ASSIST PLUS, and exchange the D-ONE tip deck for the 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, giving laboratories total flexibility to meet current and future testing demands.
• LONG 300 µl GRIPTIPS are used in all steps of this assay, simplifying the purchasing process for customers.

Assay verification

Three runs of 21 samples and 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. 5 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

Results are displayed in Tables 2-4. The standard curve for all runs displayed linearity, and all curves were within the quality parameters as defined by 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 PPC samples displayed a CV within the acceptable limit of below 25 % (Table 3).

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

Table 3: Mean coefficient of variation per plate for paired samples and PPC samples.

Remarks

• Partial plates: The supplied VIALAB programs can be adapted for partial plates, or for running samples in triplicate.
• Run report: If the ASSIST PLUS pipetting robot is connected to the PC, VIALAB programs can be started directly from the PC. After the run, a report is automatically generated, 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.