AcroPrep™ 96 Filter Plates Reduce the Presence of False Positive Results in Serological
Based Immunoassays with Luminex® xMAP® Technology
Emily Berlin, Pall Life Sciences, 600 South Wagner Road, Ann Arbor, MI 48103 • Harold Baker, Ph.D., Sonya Lowrance, Luminex Corporation, 12212 Technology Boulevard, Austin, TX 78727
Immunoassays are one of the most versatile, robust and widely used diagnostic tools in the marketplace today.
Endocrine-based and serological immunoassays are routinely used in the clinical laboratory. Thyroid Stimulating Hormone
(TSH) assays are recognized as one of the most sensitive indicators of thyroid function and are used as a primary
screening tool in the diagnosis of thyroid disease. As new generations of TSH assays have evolved, the third generation
sensitivity has increased to 0.01 – 0.03 µIU/mL, which underscores the importance of minimizing the presence of false
positives. Serological immunoassays are also used in the diagnosis of thyroid disease. In these assays, false positives can
contribute to the misdiagnosis of thyroid function and disease. This study shows that the Pall Life Sciences AcroPrep 96
filter plates with 1.2 µm Supor® membrane show a significant reduction in the presence of false positives compared to
competitive filter plates when used with the Luminex xMAP technoloy for serological immunoassays. The endocrinebased immunoassays also perform consistently with the Pall filter plates.
Background
Serological-based immunoassays are widely used as a diagnostic tool in the clinical laboratory to identify immunological
responses. Serological immunoassays detect either a specific immune response or a specific antigen to assess the
presence of autoimmune diseases. The sensitivity of serological-based immunoassays continues to evolve yielding
increasingly more sensitive assays for the detection of autoimmune diseases. The presence of a false positive response
can incorrectly indicate the presence of an antigenic response, thus leading to the misdiagnosis or treatment of the
disease. The combination of the Luminex xMAP technology and the AcroPrep 96 filter plates reliably exhibit a low
presence of false positives in both serological and endocrine-based immunoassays.
Luminex xMAP technology utilizes fluorescently encoded polystyrene microspheres to allow for multiplexing several
antigens within one sample or with other immunoassays. The xMAP technology offers numerous benefits to serological
immunoassays including: a reduction in the amount of sample required, the ability to measure the presence of multiple
antigens in a single sample, and increased testing efficiencies. The AcroPrep 96 filter plates are manufactured under a
patented process that ensures consistency, reliability, and reproducibility. The plates are a rigid, single piece construction
of 100% polypropylene, which minimizes extractables and non-specific binding. Each well is individually sealed to eliminate
the possibility of cross contamination. The Supor membrane is a hydrophilic polyethersulfone (PES) membrane that has been
optimized for low protein binding, drug compatibility, and uniform filtration rates.
Lower Level of False Postives with
AcroPrep 96 Filter Plates Than Competitive Plates
Methodology
Lot B Positive Sample
TSH (ENDOCRINE-BASED IMMUNOASSAY) PROCEDURE
xMAP Microsphere Conditions:
In the assay, there are approximately 2000 TSH antibody-coated microspheres per test point (or well) in a volume of 10 µL.
The calibrator sample is 20 µL at the indicated concentrations in a PBS buffer with 4% bovine serum albumin (BSA). The
microspheres are in a PBS buffer with 1% BSA.
Assay Conditions:
The calibrator sample and microspheres are incubated for 1 hour at room temperature. Following the incubation, 20 µL
of a biotinylated TSH antibody solution at 4 µg/mL is added to form a sandwich assay. The complex is then incubated for
30 minutes at room temperature, after which 20 µL of a 20 µg/mL Streptavidin-rPhycoerythrin (SA-rPE) solution is added.
The complex is then incubated for 15 minutes and the solution is removed from the plate under vacuum. The wells of the
AcroPrep 96 filter plate are washed twice with 100 µL PBS-1% BSA. 100 µL PBS-1% BSA is added to the plate for
reading with the Luminex LX100®. After each addition of reagents and before reading with the Luminex LX100, the
microspheres are mixed with a pipette. A total of 100 events are collected and the data is represented by the median
value of the reporter response signal from the 100 events.
Pall AcroPrep
96
Competitor M
3 µg/mL
PE-Ab
47
24
32
31
55
30
29
42
10
14
34
42
52
29
45
14
101
1313
678
1425
826
752
1118
1300
1728
692
743
905
889
770
403
895
Avg.
SD
%CV
33
13
40.97
908
399
43.89
Pall AcroPrep
96
Competitor M
4 µg/mL
PE-Ab
58
74
65
126
54
175
38
41
77
108
88
215
168
99
231
179
191
2234
972
1467
894
806
1064
1663
1302
1149
1229
1957
1012
2239
1919
1177
Avg.
SD
%CV
112
63
55.98
1330
556
41.84
Pall AcroPrep
96
Competitor M
5 µg/mL
PE-Ab
172
209
86
120
119
52
177
108
1307
1194
937
1170
455
696
478
17
1187
1740
2146
1084
4021
1628
2072
767
3584
2842
319
2657
288
656
1053
869
Avg.
SD
%CV
456
457
100.18
1682
1129
67.11
The serological immunoassays were performed with two
manufacturer’s 96 well filter plates to compare
Competitor M filter plates to Pall AcroPrep 96 filter plates.
The serological immunoassays were performed using a
single serum sample and three concentrations of
rPhycoerythrin conjugated antibody. The results from
these filter plates were read with one Luminex LX100
Instrument. The results presented are for the sixteen
individual responses, the average, the standard deviation,
and the percent coefficient of variation for the single
serum sample. The Pall Life Sciences AcroPrep 96 filter
plates exhibit a lower level of false positive results than
Competitor M filter plates.
AcroPrep 96 Filter Plates Exhibit Low Non-Specific Reactivity
with Multiple Formulations of Luminex xMAP Microspheres
Lot Number: 512
Consistent Performance in TSH (Endocrine-based Immunoassay)
Using the Pall AcroPrep 96 Filter Plates and Luminex xMAP Technology
Lot Number: 017
Lot Number: 907
Lot Number: 321
TSH
µIU/mL
AVG
AVG
AVG
0
0.003
0.01
0.03
0.1
0.3
1
3
10
30
100
300
10
13
14
19
38
69
280
751
1967
3853
8727
11245
Detection
Limit
0.003
10
11
13
17
38
56
233
1499
1850
3350
8465
8516
Detection
Limit
0.03
10
10
13
14
34
56
174
551
1854
3716
8013
10658
Detection
Limit
0.03
All Plates
AVG
10
11
13
17
37
60
229
933
1890
3640
8401
10140
Detection
Limit
0.01
Lot Number: 017
Lot Number: 907
Lot Number: 321
TSH
µIU/mL
AVG
Detection
Limit
AVG
Detection
Limit
AVG
Detection
Limit
AVG
All Plates
Detection
Limit
0
0.003
0.01
0.03
0.1
0.3
1
3
10
30
100
300
13
11
13
16
27
45
144
602
1575
2952
8071
11596
0.1
22
14
16
16
33
50
211
634
1665
3930
9184
11415
0.3
22
15
16
17
33
50
192
716
2420
4538
9592
11400
0.3
19
13
15
16
31
48
182
650
1886
3806
8949
11470
0.1
Sample
Pos 1
Pos 2
Pos 3
Neg 1
BSA
The TSH Assays were
performed with three lots of
Pall AcroPrep 96 filter
plates. The results from
these filter plates were
read with two Luminex
LX100 Instruments. The
results represent the average
of four replicates for each
calibrator concentration.
The limit of detection for
each lot of filter plates was
determined as the value at
three standard deviations
above the zero value. The
combined limit of detection
for all plates was determined
using all twelve values with
the three standard deviation
increments. The AcroPrep 96
filter plates exhibit consistent
performance across multiple
lots and instruments.
Series A
20
19
31
15
10
Series B
8220
3116
5936
17
11
Progenitor 1
126
25
40
27
21
Progenitor 2
208
27
37
34
25
SeroMAP 1
113
37
36
37
40
SeroMAP 2
160
30
36
32
28
SeroMAP 3
152
36
40
30
31
Series B
3747
1154
394
52
7
Progenitor 1
372
45
44
84
16
Progenitor 2
472
44
39
61
9
SeroMAP 1
456
30
22
62
8
SeroMAP 2
335
50
47
82
22
SeroMAP 3
538
53
47
86
24
Series B
2242
1161
876
57
7
Progenitor 1
215
33
68
86
16
Progenitor 2
259
26
64
73
11
SeroMAP 1
304
20
27
67
9
SeroMAP 2
193
39
68
99
23
SeroMAP 3
322
42
64
97
24
Series B
3720
810
1032
57
7
Progenitor 1
386
29
82
76
17
Progenitor 2
459
27
74
60
11
SeroMAP 1
524
16
37
54
11
SeroMAP 2
362
32
82
89
22
SeroMAP 3
612
34
80
84
24
Lot Number: 017
Sample
Pos 1
Pos 2
Pos 3
Neg 2
BSA
Series A
191
52
45
59
9
Lot Number: 907
Sample
Pos 1
Pos 2
Pos 3
Neg 2
BSA
Series A
134
38
75
65
9
Series A
184
28
101
58
9
Competitor M
Pall AcroPrep 96
Reduced Occurrence of False Positives
with AcroPrep 96 Filter Plates
100000
10000
Methodology
SEROLOGICAL IMMUNOASSAY PROCEDURE
xMAP Microsphere Conditions:
In the assay there are approximately 4000 microspheres per test point per microsphere type. The microsphere mixture
has 40 different microsphere sets that represent uncoupled, BSA-coated, and antigen-coated microspheres. There are
five different antigens coupled to the several types of microspheres each assayed in replicates of four. The microspheres
are in a PBS buffer with 1% BSA.
Assay Conditions:
The microspheres are in a volume of 10 µL delivered to each well with 20 µL serum sample diluted at 1:20 for an
equivalent volume of 1 µL neat serum in the well. The serum sample is diluted with PBS-4% BSA. The sample and
microspheres are incubated for 1 hour at room temperature. Following incubation, the AcroPrep 96 filter plate is filtered
under vacuum and washed twice with PBS-1% BSA. After filtration, 50 µL of buffer and 50 µL of rPE conjugated antibody
to IgG are added to the wells. The antibody is an F(ab) fragment to prevent self recognition of the constant portion of the
antibody. The complex is incubated 1 hour at room temperature, filtered and washed again with 100 µL of buffer. 100 µL
buffer is added for reading with the LX100. Mixing is done with each reagent addition.
The Serological Immunoassays were performed with four
lots of Pall AcroPrep 96 Filter Plates. The results from these
filter plates were read with one Luminex LX100 Instrument.
The responses represent the reactivity toward microspheres
that had not had proteins coupled to them to maximize the
indications of false positive “non-specific” reactivity by the
microspheres. The results represent the average of four
replicates for each the three “positive”and one “negative”
samples tested. The AcroPrep 96 filter plates show reproducible
results across multiple product lots.
Lot Number: 321
Sample
Pos 1
Pos 2
Pos 3
Neg 2
BSA
MFI Responses
Abstract
1000
100
10
1
A
B
D-1
D-2
D-3
Conclusion
Serological immunoassays play a critical role in the
clinical environment for the detection and measurement
of specific antibodies that develop in response to
exposure to disease producing/causing antigens. By
minimizing the presence of false positive results in
serological immunoassays, identification of antigenic
responses can be more accurately identified. The
AcroPrep 96 filter plates from Pall Life Sciences
consistently minimize the presence of false positive
results in serological immunoassays, thus increasing
the reliability of the assay. The combination of the
Luminex xMAP technology and the AcroPrep 96
filter plates offers a superior platform for the analysis
of serological and endocrine-based immunoassays.
Microsphere Lots
The serological immunoassays were performed with multiple lots of xMAP microspheres
in both the AcroPrep 96 and Competitor M filter plates. The results from these filter
plates were read with one Luminex LX100 Instrument. The responses represent the
reactivity toward microspheres without proteins coupled to them to maximize the
indications of false positive “non-specific” reactivity by the microspheres. The results
represent the average of four replicates for each of the three “positive” and one
“negative” samples tested. In all lots of microspheres tested, the AcroPrep filter plates
exhibited a marked reduction in non-specific reactivity than competitive plates.
Ordering Information
PALL LIFE SCIENCES
AcroPrep 96 filter plates with 1.2 µm Supor membrane,
PN 5039, 10/pkg.
LUMINEX CORPORATION
Luminex Corporation, Luminex 100™ Total System,
PN CN-L003-01.