Which endotoxin detection kit should I use?
There are a few different things you should consider before deciding which endotoxin detection kit to order. First of all, how many samples will you be testing, and how frequently? If you are going to be testing one or two samples, one time only, then you may want to consider using our Endotoxin Testing Service. If you plan to test on a regular basis, then you first need to evaluate the equipment that you currently have. (See below: “What equipment do I need to run my assay?”). If you have multiple types of equipment available for your use, consider the type of results you wish to obtain. If you must have a quantitative result, then realize that gel clot kits will not suit your needs. Gel clot tests are capable of giving only a “less than” or “more than” result. By running multiple dilutions of a sample, you may be able to narrow your sample’s EU/ml result down to a range, but gel clot kits are, at best, semi-quantitative.
Assay sensitivity is another important consideration. Our kinetic chromogenic LAL assay is our most sensitive: Kinetic-QCL® can read down to 0.005 EU/ml. PYROGENT-5000® and PyroGene® assays can both read to 0.01 EU/ml, and QCL-1000® can read to 0.1 EU/ml. PYROGENT® gel clot assays can be ordered in various sensitivities: 0.03, 0.06, 0.125, and 0.25 EU/ml. Many customers have the option to use either QCL-1000 or PYROGENT. If this is the case, decide if sensitivity or a quantitative result is more important to you.If you will be testing many samples on a frequent basis, then you may want to consider purchasing equipment and software that you do not already have. Our kinetic assays and PyroGene require less hands-on time than PYROGENT gel clot or QCL-1000, with the benefit of increased sensitivity. Lonza sells both kinetic and fluorescent microplate readers, as well as our WinKQCL® software specifically written for endotoxin-detection tests.
What Equipment Do I Need to Run My Assay?
Each endotoxin detection assay requires the use of different equipment. If you are running one of our PYROGENT Gel-Clot assays, you’ll need to have a non-circulating waterbath or a dry heat block able to heat at 37°C. If you are running our QCL-1000 assay, you’ll need a dry heat block able to heat at 37°C as well as a spectrophotometer or an ELISA reader that can read at 405-410 nm. If you are running one of our kinetic assays, Kinetic-QCL or PYROGENT-5000®, you’ll need an incubating kinetic plate reader and software. This plate reader should have a shaking function, be able to incubate at 37°C for the duration of the assay, and take kinetic readings at 405 nm (for Kinetic-QCL) or 340 nm (for PYROGENT-5000). If you are running our PyroGene assay, you’ll need an incubating fluorescence microplate reader with a 380/440 nm filter set. All of our kits require the use of a vortex mixer and standard laboratory pipettors.
Can I get an MSDS for my endotoxin detection kit and/or my accessory products?
All of our endotoxin detection products have been evaluated and deemed non-hazardous in accordance with 29 CFR 1910.1200, the Hazard Communication Standard, based on the percentage quantities of its constituents. Therefore, none of our endotoxin detection products require an MSDS.
Why should I use glassware instead of plastic?
Endotoxin adheres to plastic surfaces more strongly than to glass surfaces. Consequently, we recommend that you use only borosilicate glass dilution tubes when preparing your Control Standard Endotoxin (CSE) dilutions. In some cases, certain plastics may prove to be acceptable sample containers. While glass is still the preferred sample container material, polystyrene may be a good second choice, and polypropylene a third option. Some customers also report good results using PET. If you plan to store your sample for any period of time prior to testing, you should validate sample storage in the type of container you plan to use and verify that any endotoxin present in the sample will be recoverable after storage.
Why is it important to vortex my CSE dilutions?
Endotoxin will adhere to glass surfaces, but this can be counteracted with proper vortexing to ensure that the solution you aliquot into your reaction tubes or microplate has the proper EU/ml concentration. As our package inserts state, the CSE vial should be vigorously vortexed for 15 minutes prior to making dilutions. This should be repeated each time you use the vial, not just after reconstitution. Each CSE dilution should be vortexed for at least one minute prior to use. It is also a good idea to vortex the CSE dilutions for a few seconds immediately before use.
How do I convert from EU (Endotoxin Unit) to ng?
Depending on the source of the endotoxin, the conversion from endotoxin units to nanograms will vary. The FDA initially defined the Endotoxin Unit (EU) as the endotoxin activity of 0.2 ng of Reference Endotoxin Standard, EC-2 or 5 EU/ng. To convert the current FDA RSE, EC-6 from EU’s into ng, the conversion is 10 EU/ng. Previous versions of the BET chapter <85> in USP stated a suitable CSE has potency between 2 and 50 EU/ng.
Why should I use “matched” reagents?
You need to use “matched” reagents in order to comply with FDA requirements for endotoxin testing. Each Lonza LAL lot is tested for functionality using United States Reference Standard EC-6. We then “match” this LAL lot to a lot of our Control Standard Endotoxin (CSE) by testing in parallel with the referenced standard endotoxin (RSE). This RSE/CSE correlation assay determines the potency of that lot of CSE when used with that lot of LAL. Procedures for the RSE/CSE correlation assay are taken from the FDA’s “Guideline on the validation of the Limulus Amebocyte Lysate test as an end product endotoxin test for human and animal parenteral drugs, biological products, and medical devices.” The FDA has stated that the use of a certificate of quality from the LAL manufacturer exempts a firm from having to perform the RSE/CSE comparison on their own. Lonza makes your endotoxin testing simpler by offering kits containing “matched” reagents for all of our assay types.
How should I handle the LAL reagent?
Lonza's LAL reagent should be handled gently. After reconstitution, the contents of the vial will easily go into solution by either gently inverting or swirling the vial. Vortexing or vigorously shaking the reconstituted LAL reagent will cause it to foam. Reconstitute a few minutes prior to use, and let the vial sit on the bench top undisturbed until use. Never vortex your reaction vessel after adding the LAL.
How should I prepare my sample for testing?
There are a few ways in which your sample can be prepared before testing it. Most samples only need to be diluted before they are tested with one of our endotoxin detection kits. In order to determine how far out you should dilute your sample, you should calculate the MVD (Maximum Valid Dilution) for the sample. The MVD of a sample is its endotoxin limit in EU/ml divided by lambda, the lowest standard of the standard curve you are using (for gel-clot, lambda is the labeled sensitivity of the lysate). You should not exceed the MVD for your sample, and to ensure a margin of safety, we recommend that you do not dilute past ½ MVD whenever possible.
We recommend heat inactivation of the sample if you suspect that proteases are interfering and causing a false positive result. This can be achieved by heating a dilution of the sample at 70 degrees C for 5-15 minutes. Further dilutions can be made from the inactivated sample.
We recommend using our Beta Glucan Blocker if you suspect contamination by Beta Glucans. This contaminant can come from yeast and cellulosic materials. A common dilution/response pattern is seen in the LAL test with samples that been contaminated by Beta Glucans. This includes a negative response with concentrated samples, a positive response with increasing dilution and an eventual negative response at the highest dilution. Additionally, with kinetic methods, a synergistic response (enhancement) is frequently seen in Beta Glucan contaminated samples.
We recommend using Pyrosperse™ with samples for which endotoxin binding is a suspected source of inhibition. Some examples of samples that Pyrosperse can be used with are lipid emulsions, electrolyte solutions, normal serum albumin and plasma protein fraction.
What pH should my sample be? What should I adjust it with?
Our endotoxin detection assays are optimized to work with samples with a pH range of 6-8. If the pH of your sample falls outside of this range you can adjust it with a buffer, or HCl or NaOH. Make sure the buffer you are using is endotoxin free by running it as a sample in your assay. A good choice is Lonza's 50 mM Tris Buffer which is tested to contain less than 0.005 EU/ml.
The temperature of my refrigerator/freezer has been out of range, are my reagents ok to use?
The normal storage temperature for LAL products is 2-8°C. The improper storage of the product for a short period of time should not cause a problem with the performance of the kit as long as the components have not been reconstituted. Lyophilized components are very stable, but you should discard any reconstituted reagents. We recommend performing an initial qualification assay to confirm that the reagents still meet the performance characteristics required by the FDA. |
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