1. Multiplex immunoassay (MIA) based on xMAP© (Luminex) Technology Measles IgG performance indicators
RIVM
Centre for Infectious Disease Control
The Netherlands
Rob van Binnendijk
Fiona van der Klis
Luminex Technology xMAP®

2. Luminex xMAP technology
100 bead regions

3. Multiplex immunoassay detection
Open-architecture xMAP Technology enables multiplexing of biological tests (assays), reducing time, labor, and costs over traditional methods such as ELISA, western blotting, PCR, and traditional arrays
Antigen linked to color coded beads
Red laser reads the bead internal dye fluorescence
Green laser detects the amount of antibody bound in MFI (median fluorescent intensity)

4. Application: Immune Surveillance
vaccine coverage (IgG seropositives)
immunity in the population: correlate of protection (CoP)
> Detect gaps in immunity, groups/populations at risk

5. Multiplex Immuno Assay (MIA) : IgG quantitatively
bead
+ measles ag
(purified Edmonston, RIVM grade)
IgG in sample
detection (anti-IgG RPE)
bead
+ mumps ag (Jeryl Lynn)
+ rubella ag (HPV-77)

6. Edmonston virus produced on Vero cells (bioreactor)
concentrated by ultra filtration (1000 kD)
ultracentrifugation > disc. 20/40/70 sucrose gradient
40/70 virus fraction freeze-dried
(1-5 mg/ml)

7. Measles seroprevalence 2006/07 (Dutch cohort)
Mollema et al, Epidemiol and infect 2013

8. MIA > correlation with virus neutralizing antibodies ?
Plaque-reduction neutralization (PRN)
current standard for measuring humoral protection in measles
Edmonston (A-type) lab-strain measles virus
based on serum dilution resulting in 50% reduction of infectious centers/plaques (titer)
WHO 3rd international standard to express titers in IU/ml
estimated threshold for clinical protection 120 mIU/ml
But:
few clinical surveys support the protective threshold
PRN is labor intensive, when compared to enzyme immunoassay (EIA)
tests and serum/standard much more prone to variation
Serum dilution 4log titration 

9. * * MIA > correlation with glycoprotein-specific assays ? H F
Measles F-/H- transfected cell-lines
(de Swart et al. 1998)
serum antibody titrations
staining with FITC-conjugated anti-human IgG
FACS fluorescence analysis
sensitivity > EIA (Hartter et al. 2000)
mel/JuSo H * F *
mel/JuSo
mel/JuSo

10. MIA(IgG) correlation with anti-F, anti-H and PRN
Cohort: MMR-1 vaccinated children 4-8y (n=96)
Presera (n=96) van de kinderen (4-8 jaar) van scholen uit de Biblebelt (2013); 1x MMR gevaccineerd, vóór expositie aan mazelen (1e afname).
1. Correlatie IgG(MIA) tegen anti-H (test van Rik, = FACS-Elisa met MEL/J cellen die mazelen-H expresseren)
2. Correlatie IgG (MIA) tegen virus neutralisatie (VN): klassieke PRN test, tegen edmonston (conform 24 culture WHO protocol, 3rd WHO serum standard) F H
PRN
RIVM/EMC data 2016

11. MIA(IgG) > correlation with commercial EIAs ?
Immune status HCWs
(hospital survey)
Vaccinated birth cohorts
10..20% seronegative/indeterminate
different EIA tests, similar outcome
JMV paper 2015; selectie van 154 HCWs, geb. jaren
EIA testen; Siemens/Enzygnost (wereldwijd meest gebruikt), en nog 2 klinische testen (Liaisson, Vidas, hier niet weergegeven)
PRN: klassieke PRN test, tegen edmonston (conform 24 culture WHO protocol, 3rd WHO serum standard)
< 5% seronegative with MIA
Dorigo et al. 2015

12. Virus neutralizing antibodies
Immune status HCWs
(hospital survey)
< 1% seronegative with PRN
JMV paper 2015; selectie van 154 HCWs, geb. jaren
EIA testen; Siemens/Enzygnost (wereldwijd meest gebruikt), en nog 2 klinische testen (Liaisson, Vidas, hier niet weergegeven)
PRN: klassieke PRN test, tegen edmonston (conform 24 culture WHO protocol, 3rd WHO serum standard)
Dorigo et al. 2015

13. measles serostatus: notes and challenges
Population-wise, majority of vaccinated persons have neutralizing antibodies
Commercial EIA tests lack sensitivity, to detect protective antibodies in vaccinated persons (>10%)
Better correlation between PRN and MIA (Luminex), not absolute
Challenges:
Alternative serological tests/indicators for serological comparisons
Investigate different vaccine lots/regimens/programmes
Testing of low-titre sera and seronegatives (LLOQ)
Improve PRN testing >development high-throughput (96wells/FRN) format

14. Reducing the age of 1st MMR vaccination (6-12 months)
new studies & plans (1)
Reducing the age of 1st MMR vaccination (6-12 months)
MIA
children ‘at risk’ during latest measles epidemic in Biblebelt/The Netherlands
immunization offered between 6 and 14 months of age
clinical & immunological efficacy ( trial/study)
6-8 months
9-12 months
14m/
control n= 39 31 37
Geometric mean (IU/ml)
0,7461
2,094
2,147
<0,12 IU/ml 3
Primary immunization at 6-8 months results in decreased GMTs compared to primary immuization at 9-12 or 14 months
Preliminary data
Brinkman et al. 2016

15. new studies & plans (2)
New large population-based survey in The Netherlands 2016 (Pienter-3)
Systematic analysis of IgG (MIA) and PRN in immunized persons from different vaccinate regimes/programs/lots
Extend analysis to heterologous (wildtype) measles strains (EMC/RIVM)
Serving LabNet with high-throughput Luminex-based MMR serology
Capacity building for multiplex (MIA, FRNT) based serology for measles and mumps (EMC/RIVM)

16. Acknowledgements
Fiona van der Klis Gaby Smits Iris Brinkman Nynke Rots Jeroen Kerkhof Hinke ten Hulscher RIVM/Virology & Immunology Lab Bilthoven, The Netherlands Rik de Swart Virosciences/EMC Rotterdam, The Netherlands

17. Seroprevalence HCW study
EIA brand (manufacturer)
# tested
result
PRN <120
PRN≥120
% seropositive*
all
Enzygnost (Siemens)
154
positive
139
90.8
31/31 (100%)
56/67 (84%)
52/56 (93%)
negative 1 14
Vidas (bioMerieux)
137
89.5
55/67 (82%)
51/56 (91%) 16
Liaison (Diasorin)
136
88.9
54/67 (81%) 17
MIA (Luminex, inhouse)
149
97.4
63/67 (94%)
55/56 (98%) 4
Dorigo et al. 2015