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SimultaneousSimultaneous DetectionDetection of HIV, Hepatitis C of HIV, Hepatitis C AntibodiesAntibodies and Hepatitis B Antigen in and Hepatitis B Antigen in DriedDriedBloodBlood Spots Spots UsingUsing thethe xx--MAP Technology. MAP Technology. FollowFollow--upup of of NeonatesNeonates withwith High Antibody High Antibody
CountsCounts
Zoltan Lukacs
Hamburg University Medical Center
Institute of Clinical Chemistry and Dept. of Pediatrics
Germany
TheThe LuminexLuminex SystemSystem
Sheath fluid plate mover data recording
measurement
Dr. Z. Lukacs - University Hospital Hamburg
LabMAPLabMAP Technology Technology -- BasicsBasics
2 Lasers
532 nm, 13 mW
Reporter 1 580 nm+40 nm
633 nm, 10 mW
Classification 1 657 nm+19 nmClassification 2 730 nm+60 nm
Dr. Z. Lukacs - University Hospital Hamburg
TheThe ParadigmParadigm
96-well microtiter plate contains all analytes of interestfrom each patient in each well
Measurement
waste ?
Dr. Z. Lukacs - University Hospital Hamburg
A A MultiMulti--AnalyteAnalyte TechniqueTechnique
Dr. Z. Lukacs - University Hospital Hamburg
PickingPicking thethe right right AnalytesAnalytes
A
B
G
C
A
Dr. Z. Lukacs - University Hospital Hamburg
CalculatingCalculating ResultsResults
A
B
CD
E
F
G H
Dr. Z. Lukacs - University Hospital Hamburg
Different Different OptionsOptions
DNA Hybridisation
ELISA-type Assays
Enzyme Activity
Biochemical Studies
Dr. Z. Lukacs - University Hospital Hamburg
WhyWhy Screen Screen forfor InfectiousInfectious DiseasesDiseases ??
- Early detection may prevent further infections and may improve outcome
- Risk of HIV transmission from mother to child can be reducedfrom 15-30% to less than 2%
- True incidence of esp. HIV in many populations is still unkonwn !
It would be useful to screen „high-risk“ areas forsome infectious diseases
Dr. Z. Lukacs - University Hospital Hamburg
HIVHIV--ScreeningScreening ProtocolProtocol
punch 3 mm spots /elute with 100 µL
Filtervacuum
45 min/37 °C
add 50 µL antibody solution45 min/37 °C
add 50 µL S-RPE15 min/37 °C
add 25 µL beads
No washing steps
Total volume: 325 µLPreparation time:1h 45min
add 100 µL sheath fluid/measure
Dr. Z. Lukacs - University Hospital Hamburg
ProcessingProcessing datadata in in multimulti--analyteanalyte sessionssessions
H1 Unknown 198 873,5 102
Raw Data
Location Sample 035-HIV1 039-HIV2 Total EventsA1 Standard 46,5 889 120B1 Standard 33 1242,5 122C1 Standard 31 963 107D1 Standard 39,5 607,5 154E1 Standard 20,5 417 103F1 Standard 65,5 615 100G1 Unknown 21 518 123
Sample HIV1 Flag HIV2 Flag HepC FlagUnknown 180 134 321Unknown 200 241 256Unknown 186 213 268Unknown 132 142 198Unknown 221 168 314Unknown 142 157 187Unknown 165 164 287Unknown 138 136 249Unknown 187 174 273Unknown 164 135 276Unknown 687 Control? 469 Control? 986 Control?
Processed Data
Dr. Z. Lukacs - University Hospital Hamburg
SelectedSelected AntibodiesAntibodies
HIV 1 gp41HIV 1 gp41 HIV 1 p24HIV 1 p24 HIV 2 gp36HIV 2 gp363535 3636 3939
Hep.CHep.C NS3NS3 Hep.CHep.C NS4NS4 Hep.CHep.C corecore5050 51 51 5252
Hep.BHep.B HbsAgHbsAg73 73
Dr. Z. Lukacs - University Hospital Hamburg
ResultsResults of of NonNon--infectedinfected SubjectsSubjects
0 100 200 300 400 500
[counts]
1
2
3
4
5
6
7Su
bjec
tsHIV1gp41HIV1p24HIV2gp36HepC coreHepC NS4HepC NS3HBsAg
Dr. Z. Lukacs - University Hospital Hamburg
ResultsResults of HIVof HIV--positive positive SubjectsSubjects
0 500 1000 1500 2000 2500
[counts]
Lab. Control
Patient 1
Patient 2
Patient 3
Patient 4
Normal
Subj
ects
HIV1gp41HIV1p24HIV2gp36HepC coreHepC NS4HepC NS3HBsAg
5589
Dr. Z. Lukacs - University Hospital Hamburg
SummarySummary of of ResultsResults
At our hospital: HIV-children´s outpatient clinic
Tested : 61 children with initial antibodies
All were positive in our assay
Test results compared well withELISA andWestern Blot analysis
After storage for up to 10 years HIV antibodies were still detectable
Hepatitis B: 8 specimens were confirmed
Hepatitis C: 7 specimens were confirmed
Dr. Z. Lukacs - University Hospital Hamburg
HIV gp41 HIV gp41 –– FollowFollow--upup
0
1000
2000
3000
4000
0 100 200 300 400
Pat. OPat. PPat. Q.Pat. R
Cut-off
Age [d]
MFI
Dr. Z. Lukacs - University Hospital Hamburg
HIV p24 HIV p24 -- FollowFollow--upup
0
1000
2000
0 100 200 300 400
Pat. OPat. PPat. Q.Pat. R
Age [d]
MFI
Cut-off
Dr. Z. Lukacs - University Hospital Hamburg
CurrentlyCurrently underunder developmentdevelopment
Screening method forHereditary Fructose Intolerance
Diagnosis difficult by biochemical methods
7 Bead assay (3 frequent mutations/3 wild-type/1 nonsense)
Initial Results:
•Covers about 80% of all Caucasian patients•Fully multiplexed•Directly from dried blood spots
Dr. Z. Lukacs - University Hospital Hamburg
SummarySummary
•Luminex provides a convenient toolfor the direct multiplexed analysis of antibodies/antigens for infectiousdiseases
•Inexpensive and fast assay
•Analytes in dried blood are stable
•Simplifies follow-up procedures forfamilies with infants that may haveHIV
Dr. Z. Lukacs - University Hospital Hamburg
Acknowledgment:
Dr. Rudolf KruithofDr. Rainer GanschowAlexandra Dietrich
Dr. Z. Lukacs - University Hospital Hamburg