Antibody DetectionAntibody DetectionRelevance of cII-Relevance of cII-Specific AntibodySpecific Antibody

Andrea A. Zachary, Ph.D.

www.hopkinsmedicine.org/hla/

C’

cytotoxicity

flow cytometry

Lymphocyte-Based TestsLymphocyte-Based TestsLymphocyte-Based TestsLymphocyte-Based Tests Cytotoxicity

>35 years experience Inexpensive

Require sufficient viable lymphocytes

Reactivity strength affected by: Antigen expression Conditions of cells

Inadequate specificity Includes reactivity from non-HLA Abs Incomplete elimination of IgM

CDC: limited sensitivity

Solid Phase ImmunoassaysSolid Phase ImmunoassaysSolid Phase ImmunoassaysSolid Phase Immunoassays

Use solubilized or recombinant HLA molecules platelets transformed cell lines

Platform microtiter plates paper strips polystyrene beads

Types presence/absence specificity identification

ELISAELISAELISAELISA Pooled, solubilized Ags in microtiter

plates Sandwich assay Colorimetric determination

matrix

colored productAg

Example resultExample result: : A29>A43A29>A43 Example resultExample result: : A29>A43A29>A43

A2902

A4301

Neg

ati

ve

Posi

tive

0

10

20

30

40

50

60

70

A010

1A0201

A0301

A11

02

A2301

A2402

A250

1A2601

A2902

A3201

A3402

A3601

A4301

A6601

A6801

A74

01

B070

2B0801

B14

01

B15

01

B15

03

B15

12B15

16B18

01

B270

5B370

1B3801

B3905

B4001

B400

B410

1B4901

B50

01

B51

01

B52

01

B53

01

B55

01

Cw

08

NEG

PO

S

A0101

A0201

A0301

A1102

A2301

A2402

A2501

A2601

A2902

A3101

A3201

A3301

A3402

A3601

A4301

A6601

A6801

A6901

A7401

B0702

B0801

B1401

B1501

B1503

B1512

B1801

B2705

B3505

B3701

B3801

B3901

B4001

B4101

B4901

B5001

B5101

B5201

B5301

CW

0304

CW

0801

NE

G

PO

S

Bead AssaysBead AssaysBead AssaysBead Assays

Flow PRA bead system tested in a standard flow

cytometer

FITC anti-IgG

Alloantibody

Purified Antigen Coated Beads

FlowPRATM

Flow PRA TM Screening Beads

• Pool of different 30 Class I and 30 class II microbeads

• Class I and II beads can be separated by their different colors.

• Simultaneously detect Abs to class I & II

One Lambda Inc.

FlowPRA I & II Screening TestFlowPRA I & II Screening Test

FL1

FL2

Class I30 beads

Class I30 beads

Class II30 beads

Class II30 beads

One Lambda Inc.

FlowPRA™ Screening Test

Data Analysis

Neg. & Pos. Sera can be separated by the FL1(green) fluorescence

Pos./neg. cut-off point should be set at the end of the peak on the FL1 negative control serum histogram

One Lambda Inc.

FlowPRA™ Screening Test Data Analysis

% PRA is represented by the percentage of events shifted to the right of the cut-off point

One Lambda Inc.

Principle of FlowPRA Specific Test

1

3

4

5

6

7

8

2 2

5

FL1FL2

One Lambda Inc.

FlowPRATM I Specific Tests

FL1 Channel Shift

FL2

Cha

nnel

Shi

ft

One Lambda Inc.

Single Antigen BeadsSingle Antigen BeadsSingle Antigen BeadsSingle Antigen Beads

HOUSE JANICE AX552203.004

0 200 400 600 800 1000GOAT ANTI-HUMAN IgG FITC

1 2 3 4 5 6 7 8

GROUP#4 B BW 1__ 38 4 2__ 57 4 3__ 7 6 4__ 52 4 5__ 27 4 6__ 8 6 7__ 65 6 8__ 55 6

HOUSE JANICE AX552203.003

0 200 400 600 800 1000GOAT ANTI-HUMAN IgG FITC

1 2 3 4 5 6 7 8

GROUP#3 B BW 1__ 51 4 2__ 13 4 3__ 18 6 4__ 35 6 5 + 62 6 6 __45 6 7 __60 6 8 __44 4

Data provided by Dr. Nancy Reinsmoen, Duke University.

Bead AssaysBead AssaysBead AssaysBead Assays

Luminex system beads impregnated with varying proportions

of two dyes to permit differentiation of up to 100 beads

fluorochrome labeled antiglobulin reagent for detection of bound antibody

laser excitation of fluorochrome multiplex - can run up to 100 assays in a

single tube uses very small amounts of serum

532 nm

633 nm

PMT

APD

APDAPD

Solid Phase ImmunoassaysSolid Phase ImmunoassaysSolid Phase ImmunoassaysSolid Phase Immunoassays Increased sensitivity Increased specificity Quantifiable reactions Semi-automated: fast, high throughput

CDC: 2-4 weeks plus panel preparation time ELISA

– 111 yes/no, 1 class, 4 hours– 1 characterization - 4 hours

Luminex – 96 yes/no, cI and cII, 4 hours - 45% time saving– 96 characterizations - 4 hours -

Solid Phase ImmunoassaysSolid Phase ImmunoassaysSolid Phase ImmunoassaysSolid Phase Immunoassays Conformational changes in molecules

bound to solid phase Loss of gene expression Non-uniform detection of antibodies Panel constraints Less robust because of sensitivity Non-specific binding Increased sensitivity - clinical

interpretation

Clinical Significance of Clinical Significance of class II-specific antibodiesclass II-specific antibodies

Clinical Significance of Clinical Significance of class II-specific antibodiesclass II-specific antibodies

More than 24 cases of hyperacute or acute rejection mediated by class II-specific Abs

Multiple reports demonstrating no significance to positive B cell XM Ab specificity not determined extreme end points used

cII Ags are highly immunogenic and provoke crossreactive Abs

Incidence of Antibody to Incidence of Antibody to MismatchesMismatches

Incidence of Antibody to Incidence of Antibody to MismatchesMismatches

0

2

4

6

8

10

12

DRw51 DRw52 DRw53 DQ1 DQ2 DQ3

nu

mb

er

of

ca

se

s

negative positive

Class II CREGsClass II CREGsClass II CREGsClass II CREGs

DR1-10DR3-5-6 (DRw52)DR4-7-9 (DRw53)

DR1-2-9-10DR1-4DR5-8-w52DR10-w53

83% of patients with anti-cII had Ab to >1 Ag

Inter- and Intra-CREG AbsInter- and Intra-CREG AbsInter- and Intra-CREG AbsInter- and Intra-CREG Abs

27

0

20

40

60

80

100

per

cen

t

DRw52 DRw53 DR1, 2, 10

27 3454

85

8531

Inter-CREG AB

Intra-CREG Ab

Clinical Significance of Clinical Significance of class II-specific antibodiesclass II-specific antibodies

Clinical Significance of Clinical Significance of class II-specific antibodiesclass II-specific antibodies

16 patients in desensitization protocol (plasmapheresis + low dose CMVIg)

All had Ab to donor cII but not cI 11 with no cI-specific Ab 3 with no cI mismatches

Patients with Ab to Donor Patients with Ab to Donor cIIcII

Patients with Ab to Donor Patients with Ab to Donor cIIcII

Ab at or After Tx

AMR StatisticsNone

Flow XM +

CDC XM +

5 0 0 1 2 = 7.4

P<0.0010 7 3 9

ConclusionsConclusionsConclusionsConclusions

Current technology permits rapid, accurate, sensitive detection and characterization for HLA-specific antibodies.

Antibody to donor HLA (class I or class II) are a risk factor for graft dysfunction, regardless of titer, if untreated.