Introduction
Endotoxin detection remains an extremely important aspect of research and treatments that are derived from expression in gram-negative bacteria1. Because of the significant negative reaction that can stem from even small amounts of endotoxin in a preparation, detection and subsequent elimination of endotoxin is vital.
The most popular means of detecting endotoxin are varieties of the limulus amebocyte lysate (LAL) assay. The limulus amebocytes are obtained from the blood of the horseshoe crab. Although much care is taken to extract this reagent carefully and safely return the crabs to the ocean, the long-term viability of this approach is questionable. The PyroGeneTM method, based on recombinant factor C (rFC) obviates the need for horseshoe crab blood.
One of the features of the new CLARIOstar Plus is the enhanced dynamic range (EDR) capability. EDR allows you to detect both very high and low signals on the same plate and simplifies assay setup especially for kinetic assays. Gathering data in a kinetic manner and using EDR, we found that the PyroGeneTM fluorescent assay can potentially be detected with equivalent sensitivity as the standard PyroGeneTM protocol in far less time, as quickly as the QCL-1000TM (LAL) protocol.
Assay Principle
Factor C is a protease whose normal function is to serve as an initiator of the limulus coagulation cascade upon exposure to lipopolysaccharide such as endotoxin2. Its natural substrate is factor B and knowledge of the cleavage site in factor B has enabled production of substrates that can be detected in in vitro assays.
Figure 1 shows the principle on which the PyroGeneTM assay is based. Binding of endotoxin to rFC results in an active form of the enzyme. This can then cleave a fluorogenic substrate, releasing the detectable fluorophore.
Materials & Methods
- Black, 96-well, clear bottom, non-pyrogenic microplates (Corning)
- CLARIOstar Plus (BMG LABTECH)
- PyroGeneTM Recombinant Factor C Endpoint Assay (LONZA)
- Endotoxin-free pipette tips and tubes (LONZA)
Experimental procedure
A 20 IU/mL standard was created as described in kit instructions. Similarly, standards of the indicated concentrations were made by dilution of the 20 IU/mL standard to span concentrations from 10 IU to 0.0005 IU/mL. Replicates of each of the standards (n=5), and replicates of the pyrogen-free water blank (n=12) were added to a microplate at a volume of 100 µl. The microplate was pre-incubated at 37 °C for at least 10 minutes after which 100 µl of a detection reagent mix, prepared as indicated in kit instructions, was added. The fluorescence produced in the plate was read on the CLARIOstar Plus as follows:
Instrument Settings
|
Optic settings |
Fluorescence Intensity, plate mode kinetic | |
| Monochromator settings | ||
| Excitation | 371-15 | |
| Dichroic | Auto 407.8 | |
| Emission | 447-20 | |
| Gain obtained by | EDR | |
| Focal height | Autofocus (7mm) | |
|
General settings |
Settling time |
0.5 s |
|
Incubation |
37 °C |
|
| Kinetic |
Number of cycles |
13 |
|
Cycle time |
300 s |
|
|
Number of flashes |
20 |
|
Results & Discussion
The typical PyroGeneTM test employs an initial reading immediately (t=0) followed by a second reading after a 1-hour incubation. The t=0 reading provides a baseline that can be subtracted from the result of incubation. We sought to discover how quickly we could distinguish between the various concentrations of endotoxin using EDR on the CLARIOstar Plus. To this end, we read the plate at 5-minute intervals over the usual 1-hour time course. The results of this experiment, presented in Figure 2, show that higher concentrations of endotoxin exhibit a robust increase in fluorescence, detectable in as little as 10 minutes EDR allows for a very wide range of fluorescent intensities to be detected in a single plate.
Due to this fact, a typical scale can sometimes obscure relatively small changes in signal. Figure 3 shows that this is true for the response of the PyroGeneTM test. Significant, lower intensity signal changes are seen for the 2 lowest concentrations of endotoxin employed.
To analyze the results of PyroGeneTM tests a log-log transformation of the data is employed to enable prediction of unknowns. Figure 4 is typical for our results and compares the linear fit for the data under this transformation at 15, 20, and 60 minutes. The data conforms very well in this transformed fit, with R2 values of 1 for all times shown.
We next sought to determine the reproducibility of the assay, especially at early time points. To this end, the approach described was repeated to obtain 4 assay replicates. The results shown in table 1 provide %CV range from these experiments. The %CV at 15 and 20 minutes are comparable to those achieved at the 60 minutes and are certainly within the variability specification for this assay by LONZA.
Table 1 Comparison of variability for PyroGene™ assay
|
|
% CV Ranges at time (minutes) |
||
|
[endotoxin] |
15 | 20 | 60 |
|
.005 IU |
3.4-19.3 | 3.5-14.1 | 6.5-14.4 |
|
.05 IU |
4.5-23.7 | 3.1-24 | 4.3-13.8 |
|
.5 IU |
7-21 | 5.8-18.8 | 4.3-14.9 |
|
5 IU |
7.7-20.9 | 5.9-19 | 3-11.9 |
|
10 IU |
3.2-18.4 | 2.7-16.3 | 2.4-7.5 |
Conclusion
The EDR function of the CLARIOstar Plus makes sensitive detection easy. Here we show how this can be advantageous, allowing the PyroGeneTM assay from LONZA to be performed in just 15 minutes without loss of sensitivity.*
References
-
Su, W. & Ding, X. (2015). J. Lab. Auto. 20: 354-364. DOI: 10.1177/2211068215572136.
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Muta, T., et al. (1991) J. Biol. Chem. 266: 6654-6661
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Das, A. P., et al. (2014) Biosens. Bioelectron 51: 62-75. DOI: 10.1016/j.bios.2013.07.020
*Note: Testing conducted for this study was performed outside of the assay instructions defined in the PyroGene™ rFC Assay package insert. Lonza does not make guarantee of performance for any test procedures or assay performance specifications that are not explicitly stated in the product instructions. End-users are responsible for validating any deviation to the procedures outlined in the product package insert.