Purpose
With the introduction of pioneering technology for bacterial endotoxins testing (BET), it is critical to demonstrate equivalency across platforms. The Sievers Eclipse Ratio Verification Test was developed to confirm that the 1:1 ratio of sample to lysate typically used in a 96-well microplate is ever-present when utilizing the Eclipse BET microplate.
Providing a 1:1 ratio each time supports a consistent biochemical interaction between samples and Limulus Amoebocyte Lysate (LAL). Despite a reduction in volume when utilizing the Sievers Eclipse platform, the ratio is still the same, and end users can rest assured that endotoxin values for the samples and standards are equivalent and legitimate.
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Background and importance
The technique, known as kinetic chromogenic BET with a 96-well microplate, requires mixing of standards and samples with reagent in each well and witnessing the changing color over a period of time. Precise control over the amount of sample against the amount of lysate is a key factor for success. USP stipulates that the lysate manufacturer’s instructions should be adhered to for appropriate use (IFU) with respect to specific test parameters including incubation time, volume ratios, or pH. The manufacturer’s IFUs state how to best acquire the correct volume ratio of sample to reagent in two ways:
(1) Directly stating that the ratio of sample to reagent should be 1:1
(2) Instructing end users to add 100 µL of LAL reagent water blanks, endotoxin standards, product samples, and positive product controls, then adding 100 µL of LAL to all utilized wells.
By instructing users that the ratio of sample to reagent should be 1:1, one IFU advises on a total well volume of 100 µL, rather than the typical 200 µL, indicating that it is ideal for the greatest sensitivity. This IFU shows that the 1:1 ratio is the major element in this assay, not the overall well volume. As long as there is a 1:1 ratio of sample to lysate present in each well, the reaction will progress accurately, and the results yielded will be equivalent.
If there is a disproportionate ratio, as in the exact amount of sample to reagent is no longer correct, it may have a considerable impact on reaction kinetics and the total assay results. Regardless of the overall well volume, the reaction could happen faster, generating a shorter onset time, and consequently higher endotoxin concentrations. Conversely, the resulting reaction could produce a slower onset time and lower endotoxin concentrations. Having a ratio that is inconsistent could also influence the natural buffering capabilities that the lysate provides when combined with the sample. Without a precise 1:1 ratio, the pH of the reaction mixture can be modified beyond the recommended range of 6-8 and impact the overall kinetics of the assay.
The 1:1 Ratio Verification Test was designed to show that the precise ratio of lysate and sample is achievable on the Sievers Eclipse microplate in each one of the 104 wells. To conclude the test, the user will require a new Sievers Eclipse microplate and the Sievers Eclipse 1:1 Ratio Verification Kit (STD 85000- 01), which is comprised of a water vial and a dye vial. The Eclipse software guides the user through the setup and injection of water and dye into the relative segments and the LAL port prior to initiating analysis. The Eclipse microplate then moves through the same protocol as standard analysis, but because the 1:1 Ratio Verification Test does not depend on a kinetic enzymatic reaction, there is a reduction in the total testing time.
Report details for the 1:1 Ratio Verification Test
When completion of the analysis has occurred, a report is filed in the 1:1 Ratio Results tab of the Eclipse software. As well as the general information registered before running the test (e.g., Eclipse microplate information, analyzer serial number, and 1:1 Ratio Verification Sample information), the report illustrates the average optical density for each single one of the 104 optical wells. In the results section of the report, there is an overall average optical density for the dye portion of the plate and also an overall average optical density for the water portion of the plate. Two dyes are mixed on one half of the plate, and one dye combined with water on the other half of the plate generating each of these two averages. Equation 1 (below) is used to acquire an overall ratio value that stipulates the success of the mixing. This is suggestive of how the sample and LAL will be combined during conventional analysis.
Equation 1.
Dye Average
———————- = Ratio Value
Water Average
In the wells where two dyes are mixed together, an optimal ratio would be 1 (Dye Average). In the wells where dye is mixed with water, the dilution of the dye to half of its original concentration means that the ideal ratio is 0.5 (Water Average). When dividing these averages, the resulting optimal Ratio Value is 2, signifying the precise amounts of both dye and water that reached the optical wells. A ratio between 1.90-2.10 is deemed valid for this test and will not influence the recovery of endotoxin or overall kinetic curve.
Conclusion
The precision-crafted Sievers Eclipse microplate is a microfluidic liquid handling device that leverages the use of metering chambers, in conjunction with consistent channel geometry and motion, to simultaneously supply the exact same amount of sample and lysate to the optical wells. It is this precision liquid handling with the Eclipse microplate that guarantees to achieve the crucial 1:1 sample to lysate ratio across all 104 optical wells for each single assay.
While a 1:1 ratio is either stated by the lysate manufacturer’s IFUs, or it is stated to add a specific amount of sample and reagent, it is not always 200 µL. The volumes advised are generally set to help enhance the precision due to a prolonged path length or to facilitate more accurate pipetting by utilizing a larger volume. They are also within the standard well working volume of 75-200 µL that is advised for the size of the wells on a conventional 96-well microplate.
“In order to have an efficient and realistic measurement, the lowest volume recommended for a microplate well is generally >1/3 of the maximum volume of the well”.1 This results in the key factor, a 1:1 ratio of sample to lysate in each single well, being achieved every time, rather than just the total well volume. The 1:1 Ratio Verification Test on the Sievers Eclipse BET Platform demonstrates that even with a considerably smaller total well volume, the key 1:1 ratio is maintained in each optical well, every single time. With the precise ratio ever-present, the end user can rest assured that the biochemistry is equivalent. It is always advised that this verification test be conducted yearly by a SUEZ representative or Certified Field Service Engineer.
References
- Pusterla, Tobias, Ph.D. “Which is the best microplate for my assay?” BMG Labtech, 2018 May 30. https://www.bmglabtech.com/which-isthe-best-microplate-for-my-assay
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