UCP-LFA for IFN - Seventh EDCTP Forum

Field-evaluation of a UCP-LF assay for detection of
Cellular & Humoral Immunity
against mycobacteria
Annemieke Geluk
Dept. Infectious Diseases
Leiden University Medical Center
7th EDCTP Forum
Berlin, 1st July 2014
Leprosy
 Infectious disease caused by Mycobacterium leprae
 Major threat in developing countries (WHO)
 Ranks 2nd behind Mtb as human mycobacterial disease
 Affects skin & nerves
 Treatable (MDT) if detected early!!!
 Results in severe, life-long disabilities and deformities
due to delayed diagnosis
(Facing the) Challenges in Leprosy
Leprosy Disease Spectrum
CMI
T cells
HMI
Ab
TT
BT
BB
BL
LL
Paucibacillary (PB) - Multibacillary (MB)
New Test Development
• Develop easy-to-use field test for early detection of leprosy
before clinical signs
• Based on detection of multiple Biomarkers (CMI & HMI)
• Applicable in field setting
UCP-based assay for diagnostics
• UCP = Up-Converting Phosphor
• Applications in diagnostics for HIV, TB, Schistosomiasis
• UCP particles contain a crystal structure
• The structure contains rare earth metals that absorb IR light
and emit in visible spectrum
• Low background (rare earth metals)
• UCP compounds are chemically stable (long-shelf life)
• Do not bleach: transport without cold chain =>
allowing re-analysis in reference lab
UCP-LFA for IFN-
1.
Incubate sample with UCP-labeled, anti-IFN- mAb
UCP-MH
IFN-
UCP-MH
IFN-
UCP-MH
IFN-
UCP-MH
IFN-
2.
Flow over anti-human IFN- mAb and control (GAM) strip
UCP-MH
IFN-
Mouse anti human IFN- mAb2
UCP-MH
IFN-
Flow Control (GAM)
WBA (tool)
24h WBA
Similar to Quantiferon
lateral flow
IP-10 detection by UCP-LFA
• Produced in high levels (100-fold)
• Not influenced by low CD4 counts
• Can indicate M. leprae exposure
• Biomarker type 1 leprosy reactions
• Biomarker for TB infection (Ruhwald et al.)
IP-10 kinetics
M.leprae LP
ML2478 LP
15,000
PHA LP
15,000
100,000
80,000
10,000
60,000
10,000
5,000
4,000
IP-10 (pg/ml)
IP-10 (pg/ml)
IP-10 (pg/ml)
40,000
5,000
4,000
20,000
6,000
4,000
2,000
2,000
2,000
0
0
t=1
t=4
t=6
time (hours)
t=24
0
t=1
M.leprae EC
t=4
t=6
time (hours)
t=24
t=1
ML2478 EC
15,000
t=4
t=6
time (hours)
t=24
PHA EC
15,000
100,000
80,000
10,000
60,000
10,000
5,000
4,000
IP-10 (pg/ml)
IP-10 (pg/ml)
IP-10 (pg/ml)
40,000
5,000
4,000
20,000
6,000
4,000
2,000
2,000
2,000
0
0
t=1
A
t=4
t=6
time (hours)
t=24
0
t=1
t=4
t=6
time (hours)
t=24
t=1
t=4
t=6
time (hours)
t=24
Bobosha et al, 2014
IL-10 & IP-10/IL-10 ratio
unstimulated
plasma
stimulated
WBA plasma
1200
1000
800
600
p< 0.0001
p=0.039
p=0.051
ratio IP-10/IL-10
IL-10 (pg/ml)
IL-10
500
400
300
200
4500
3500
2500
1500
500
500
400
300
200
100
100
0
0
-
-
PHA
PHA
Mleprae Mleprae ML2478 ML2478
LP
EC
= EC
= LP
Bobosha et al, 2014
Correlation ELISA vs UCP-LFA
(single; dry format)
Ethiopia Leprosy patients & EC
anti-PGL-I
ELISA (sample ID)
20
10
5
10
0
10
20
30
UCP (sample ID)
40
30
20
10
0
0
IL-10
40
15
30
ELISA (sample ID)
ELISA (sample ID)
20
IP-10
40
0
0
5
10
15
UCP (sample ID)
R2 = 0,790
R2 = 0,689
n = 39
n = 19
20
0
10
20
30
UCP (sample ID)
40
R2 = 0,735
n = 30
Bobosha et al, 2014
Handheld vs 96-microtiter
Pearson correlation
Spearman ranking
IP-10
Packard Scanner (sample ID)
Packard Scanner (ratio)
IP-10
400
5
4
3
2
1
R2= 0.941
300
200
100
0
0
0
1
2
3
4
5
0
UCP-Quant Scanner (ratio)
Packard Scanner (sample ID)
Packard Scanner (ratio)
300
400
anti-PGL-I
400
4
3
2
1
200
UCP-Quant Scanner (sample ID)
anti-PGL-I
5
100
R2= 0.967
0
300
200
100
0
0
1
2
3
ESE Scanner (ratio)
4
5
0
100
200
300
UCP-Quant Scanner (sample ID)
400
Bobosha et al, 2014
Single vs multiplex UCP-LFA
Pearson correlation
IP-10
200
single UCP (sample ID)
2.5
2.0
1.5
1.0
single UCP (ratio)
Spearman ranking
0.5
0.4
0.3
0.2
0.1
150
100
50
R2 = 0.897
0.0
0.0
IP-10
0
0.1
0.2
0.3
0.4
0.5 1.0 1.5 2.0 2.5
0
multiplex UCP (ratio)
8
50
100
150
200
multiplex UCP (sample ID)
150
anti-PGL-I
anti-PGL-I
6
single UCP (sample ID)
4
single UCP (ratio)
2
0.5
0.4
0.3
0.2
0.1
50
R2 = 0.961
0
0.0
0.0
100
0.1
0.2
0.3
0.4
0.5
multiplex UCP (ratio)
2
4
6
8
0
50
100
150
multiplex UCP (sample ID)
Bobosha et al, 2014
Summary
• Detection of IP-10 levels in stimulated samples allowed a reduction of the
WBA from 24h to 6h.
• IP-10/IL-10 ratios in unstimulated plasma can discriminate between leprosy
patients and EC, thereby allowing identification of infection.
• Dry-format UCP-LFAs are low-tech assays allowing multiplex detection of
relevant cytokines and antibodies in response to M. leprae in the field.
Contributions
LUMC, NL
Annemieke Geluk
Jolien vd Ploeg-Schip
Susan van den Eeden
Kees Franken
Elisa Tjon Kon Fat
Claudia de Dood
Tom Ottenhoff
Paul Corstjens
Funding
Q.M. GastmannWichers Foundation
Armauer Hansen Research
Institute (AHRI)
Kidist Bobosha
Yonas Bekele
CSU, USA
John Spencer

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