Pharma&Biotech
Stepped Neutral Density Plate
Translated versions available at www.lonza.com
Content
Section
Page No.
1 Intended Use 2
1 Background and Test Principle 2
1 Material 3
2Storage 4
2 Performance Certification and Cleaning 4
2Materials and Equipment Not Provided
5
2 Explanation of Test 5
3 Test Procedure 6
3 Performance Characteristics
7
Intended Use
The stepped neutral density (SND) plate is intended for use in the
validation of the BioTek® ELx808™ Microplate Reader (Cat. No. 25-315
or 25-315S). The SND plate may also be used in the validation of other
microplate readers at the user’s discretion.
Background and Test Principle
The Kinetic-QCL™ and PYROGENT™-5000 Endotoxin Detection Assays
require a 96-well microplate reader that is capable of monitoring a
change in optical density (OD) at 405 nm and 340 nm, respectively.
Providing evidence that the microplate reader correctly monitors
changes in OD is an important part of the microplate reader validation
process that can be completed with the SND plate.
2
Material
The SND plate contains six regions of linearly increasing OD in a dry
plate format as shown in Figure 1. Each region consists of two columns
of microplate wells, and the OD is uniform within each region. The six
neutral density filters in the SND plate are composed of a thin metal
film that is uniformly deposited on a fused silica substrate that is
transparent to UV light. The first filter region is uncoated substrate that
is used as a blank, and the average OD of the first two columns of wells
is typically subtracted from the OD values found on the remaining five
neutral density filter regions.
Figure 1. Stepped Neutral Density Plate
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Important: Read Entire Brochure Before Performing Test
3
1
Storage
Materials and Equipment NOT Provided
The SND plate should be placed inside the provided plastic bag and
stored in the foam cushioned case when not in use. Store at room
temperature and use care when handling.
1. WinKQCL™ Software.
2.BioTek® ELx808™ microplate reader or other 96-well microplate
reader.
Performance Certification and Cleaning
Each filter lot is quality tested after production using a (National Institute
of Standards and Technology) NIST-calibrated spectrophotometer to
determine the optical density of the filter by scanning between 300 nm
and 500 nm. The performance certification is done in a calibrated
microplate reader at both 405 nm and 340 nm to ensure that each filter
meets uniformity specifications and that the increase in optical density
from region to region is linear. Annual cleaning and recertification of
the SND plate is recommended. Contact Scientific Support for details
on this procedure, which will include verifying filter conformity to
the original manufacturing specifications using a NIST-calibrated
spectrophotometer.
Explanation of Test
Validation of the linearity of the optical density measurement is
accomplished through the use of WinKQCL™ Software. The program
is already configured to read the SND plate and analyze the data. The
WinKQCL™ Software will interpret the plate as containing a blank and
five standards arranged in columns, with each of these six regions
consisting of two columns of eight wells. After the plate is read, the
program will calculate a linear curve using each of the five standards
after subtracting the blank values from each region. The variation within
each region will also be calculated.
If the SND plate is stored in the provided plastic bag and handled
properly, only surface contamination of the plate by environmental
dust will occur. In most cases, it is best to leave minor debris on the
surface as long as the performance of the plate is not affected. To
dislodge debris from the surface of the filters, the use of oil-free dry air
or nitrogen under moderate pressure is recommended. Should a more
thorough cleaning be required, contact Scientific Support to arrange
for a thorough cleaning.
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5
2
Test Procedure
Performance Characteristics
To begin the test, insert the plate into the microplate reader carrier
with the label on the left side facing up as shown in Figure 1. Using the
OD Validation feature of the WinKQCL™ Software, the SND plate can be
read and analyzed at either 405 nm or 340 nm. Consult the WinKQCL™
Software Manual for detailed instructions regarding this software
feature.
The microplate reader is interpreting increasing OD values linearly if
the analysis of the SND plate conforms to the following specifications
for 405 nm and 340 nm. Note that while the plate may be used at other
wavelengths and in other instruments, these specifications have been
developed for the 405 nm and 340 nm filters of the BioTek® ELx808™
Microplate Reader. If used in another instrument, the slope of the
resulting standard curve may vary somewhat from that specified below.
For use without the WinKQCL™ Software, it will be necessary to employ
other means to calculate the standard curve and coefficient of variation
(CV) of each of the zones. The standard curve should be generated based
on a blank and five standards (S1 to S5) with doubling concentrations
of 0.0625, 0.125, 0.250, 0.500 and 1.000 in vertical replicates of 16
as shown in Figure 1.
Linearity
Correlation coefficient
r ≥ 0.990
Reproducibility
CV for each standard
S1 0.0625 < 10.00%
S2 0.125 < 5.00%
S3 0.250 < 2.00%
S4 0.500 < 2.00%
S5 1.000 < 2.00%
3
Slope
Standard curve
6
1.440–1.760
7
Notes
Notes
3
8
9
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