simultaneous detection of hiv, hepatitis c antibodies and ... › conferences › proceedings ›...

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