SoGAT 2005

Donor screening for parvovirus B19 antibodies:

Reducing or eliminating the risk of transmission

SoGAT 2005

Gordon ElliottBiotrin

93 The RiseMount Merrion

DublinIreland

SoGAT 2005

TMB

VP2 HRP

OD450

IgGELISA

TMBCE MarkedFDA Approved

Intact Viral particles are essential for 100% detection of B19.

VP1/2 antigens expressed in E.Coli do not form particles and give 10-20% false negative IgG results.

Electron Micrograph of Biotrin VP2 Capsids

SoGAT 2005

TMB

VP2 HRP

OD450

IgGELISA

TMBCE MarkedFDA Approved

VP2HRP TMB

OD450

IgMELISA

CE MarkedFDA Approved

Virus HRPTMB

OD450

AntigenELISA

Research assay

SoGAT 2005

Vireamic donations - antibody profiles:

70 Vireamic (>10e6 IU / ml) samples collected over 12- 18 month period

•Biotrin examined the B19 IgG and IgM levels

IgG and IgM EIA Index Value Distribution

0

1

2

3

4

5

6

7

8

9

10

5678910111213

B19 IU/ ml (log10)

IgM

IgG

SoGAT 2005

IgG EIA Index Value Distribution

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

56789101112

B19 IU/ml (log10)

Ind

ex V

alu

e

IgG

SoGAT 2005

Specificity of the assay:

To evaluate integrity of positive results at the low end of the range - <2.5 index

•In a panel of 500 blood donors, 350 (70%) were IgG positive.•18 samples (3.5%) were ‘very low pos’, index 1.1 to 2.5•These 18 samples were tested by confirmatory competitive (VP2) assay

SoGAT 2005

Specificity of the assay:

Index Status Comp Index Confirmed StatusLP1 1.2 Pos 0.2 PosLP2 1.3 Pos 0.2 PosLP3 1.4 Pos 0.07 PosLP4 1.5 Pos 0.1 PosLP5 1.6 Pos 0.06 PosLP6 1.7 Pos 0.1 PosLP7 1.7 Pos 0.1 PosLP8 1.7 Pos 0.06 PosLP9 1.8 Pos 0.1 PosLP10 1.9 Pos 0.1 PosLP11 1.9 Pos 0.2 PosLP12 2 Pos 0.07 PosLP13 2 Pos 0.6 PosLP14 2.3 Pos 0.4 PosLP15 2.3 Pos 0.2 PosLP16 2.3 Pos 0.2 PosLP17 2.5 Pos 0.3 PosLP18 2.5 Pos 0.1 Pos

SoGAT 2005

Effectiveness of IgG donor screening:

•Normal donor population (Netherlands) 80% are IgG reactive

•Of all vireamic specimens detected by plasma PCR screen process (>10e6 IU ml), in a 12-18 month period, 0% true IgG positives (n=70)

•Rate of parvo IgG in normal donors is possibly at least 80 times rate in vireamic donors

•Typically 1 in 10,000 fresh blood product units donated are vireamic(>10e5 IU / ml) (greater in epidemic)

•Consider that with a 1 time IgG screen at the time of donation, the rate of vireamic donations may be better than 1 in 800,000

(Allowing for 1 false positive in this case, 1.43% are ‘apparent reactive’rate of vireamic donations would be of the order of > 1 in 560,000)

SoGAT 2005

Current Sanquin Approach: The two-time testing model is clearly successful in reducing, if not eliminating, high vireamic donations.

•Main drawback is requirement for time gap in two-time testing

•One time screening using combined tests maybe possible:

1) IgG+ / IgM- donors (zero of 70 viraemics tested)

2) IgG+ / Parvo Ag- donors

3) IgG+ / PCR low donors

Studies ongoing

SoGAT 2005

Use of antigen in Parvovirus IgG ELISAs

The Biotrin assay is based on G1 (B19) VP2 Capsid:

• Is VP1 and VP2 required for detection of all positives?

•Does the Biotrin assay work for Genotype 2 (A6) and Genotype 3 (V9)?

Collaboration with Prof. JP Allain, Cambridge Transfusion Medicine

SoGAT 2005

Servant et al, 2002Servant et al, 2002

Genotype 1 (B19) VP2Genotype 1 (B19) VP1/2Genotype 3 (V9) VP2Genotype 3 (V9) VP1/2

‘CAM 1’ ‘CAM2’ ‘CAM3’ ‘CAM4’

CODE 1

CAMBRIDGEBIOTRIN

CODE 2

Test 500 donors from G1 (B19) region (UK)Test 500 donors from G3 (V9) region (Ghana)

SoGAT 2005

No % No % No % No %Positives 98 71 96 70 98 71 98 71Negatives 39 28 41 30 40 29 40 29Equivocal 1 1 1 1 0 0 0 0Total 138 100 138 100 138 100 138 100

CAM4CAM3CAM2CAM1

UK Donor Population - Antigen Reactivity

0

20

40

60

80

100

120

CAM1 CAM2 CAM3 CAM4

Antigen

N

Positive

Negative

Equivocal

138 UK Donors tested (to date) using several capsid antigens

SoGAT 2005

500 Ghanaian Donors:

> 100 tested to date

•Different index value distribution compared to European populations

•Lower Index Values overall

•But no difference in results between antigens.

Testing of Ghanaian Children < 5 yrs demonstrated that G3 Antisera reacts with Biotrin’s G1 antigen as well as with experimental G3 antigen.

G3 derived antibodies are detected as well as G1 derived antibodies using the current assay.

SoGAT 2005

An Antigen-Capture EIA for the detection of Parvovirus B19 in human plasma.

•The first EIA for B19 Virus Detection.

• Employs a classic “sandwich” EIA format.

• Sensitivity of approximately 5 x 107 IU / ml.

• High specificity, no false-positive results in over 500 normal human plasmas tested.

• Capability to detect virus in the presence of high levels of anti-B19 IgM and / or IgG.

• Detection of variants e.g. V9

SoGAT 2005

B19 Viremic Sample qPCR: geq/ml* Index / Virus EIAStatus

Sample 1 :Bx 71 3X108 1.98 Positive Bx 71 3X107 0.43 NegativeSample2: Bx 72 4X108 3.11 Positive Bx 72 4X107 0.96 EquivocalSample3 Bx 73 4X108 2.96 Positive Bx 73 4X107 1.01 Positive

SoGAT 2005

Sanquin:Theo CuypersHarry BosMarco Koppelman

Cambridge:JP AllainDaniel CandottiArmen Parsyan

Biotrin:Shane KerrAmanda Corcoran