Application Note

Comparing performance of SpectraMax absorbance microplate readers using an endotoxin assay

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Materials

Methods

Both readers were heated to 37ºC prior to setting up the assay.

Reconstitution of reagents

Standard curve

One standard curve plate was set up for each reader:

Parameter
Settings
Read mode
Absorbance (ABS)
Read type
Kinetic
Wavelength
340 nm
Plate type
96-well standard clear bottom
Timing

Total run time: 01:40:00

Interval: 00:01:00

More settings
Carriage speed: Slow

Table 1. Instrument settings for kinetic turbidimetric assay on the SpectraMax ABS Plus and SpectraMax Plus 384 readers. A kinetic chromogenic LAL protocol in SoftMax Pro Software contains optimal settings and automates the analysis of results.

Uniformity test plate

Using the Data Reduction dialog in SoftMax Pro Software, the following parameters were used:

Average onset times were calculated by the software and plotted vs. endotoxin concentration.

Comparison of endotoxin data from both readers

Figure 1 shows the standard curves obtained from the SpectraMax ABS Plus and Plus 384 readers. Both standard curves had r2 values greater than 0.99, and for each standard (n = 3) the %CV values were less than 7.

Endotoxin standard curves

Figure 1: Endotoxin standard curves on SpectraMax microplate readers, plotted using a log-log curve fit. For SpectraMax ABS Plus reader, r2 = 0.996 (n = 3) ; for SpectraMax Plus 384 reader, r2 = 0.997 (n = 3).

Table 2 summarizes the results for the uniformity test. Here, the CVs for each plate were less than 3, and the difference between mean onset time for the plates run on the two readers was only 7%.

Instrument
Endotoxin, EU/mL
Mean value
SD
CV
SpectraMax ABS Plus
0.1(ABS)
1060
27
2.5
SpectraMax Plus384
0.1(ABS)
985
23
2.4

Table 2. Uniformity between SpectraMax microplate readers. A 7% difference in mean onset value for 0.1 EU/mL standard was observed between SpectraMax ABS Plus and SpectraMax Plus 384 readers.

Together, these results demonstrate that both SpectraMax ABS Plus and SpectraMax Plus 384 readers will deliver equivalent results: highly linear standard curves enable accurate endotoxin quantitation, and low cross-plate %CVs assure uniformity of results.

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