Laboratory orientation

Ping Chin Lai

HLA – Human Leucocyte Antigen

Equivalent to Major Histocompatibility Complex (MHC) in mouse

On the short arm of chromosome 6 Highly polymorphic Responsible for recognition of cells as self or f

oreign. They can present foreign peptides to T-cell receptor in order to initiate immune response.

T-Cell

Variable Region

Constant Region

Antigen Peptide

MHC

Peptide Binding Groove

MHC II MHC I

Human Leukocyte Antigens (HLA)

2M

All Nucleated CellsB-Cells, Macrophages

HLA Typing in Laboratory

Microlymphocytoxicity assays

HLA Class I

Mixed lymphocyte culture

HLA Class II

Microlymphocytotoxicity test (微量淋巴球細胞毒殺試驗 )

原理 補體依賴細胞毒殺反應 (CDC) – 1964 Terasaki

藉由具特異性抗血清與淋巴球細胞膜上的相對 HLA抗原結合 ,活化補體 (新鮮冷凍兔血清 ),造成細胞膜損傷 ,致使活性染料進入細胞膜內

Serum

+

Target

Lymphocytes

Rabbit

Complement

Eosin/

Flurorescent

Satin Formalin

Read

Cell

Lysis

已知

未知

DNA based Typing Methods

SSP

RFLPrev. SSOP

SSOP

SBT

PCR

PCR-SSP Method

PCR-SSP (Sequence Specific Primer)

1. DNA Extraction

2. PCR using SSP’s

3. Agarose Gel Electrophoresis

Characteristic Typing Occurs During Amplif

ication Establish linkages between

polymorphisms Throughput Depends on the

# of thermocyclers Rapid

MatchedAmplification

NoAmplificationMismatched

PCR-SSP Principle

Positive Reaction

G G G G T G C C C T A A A A

A T T T T

G G G G T

C C C C A C G G G A T T T T

A GC

5’

3’

3’

5’

5’

230

Sequence-Specific Amplification

PCR-SSP Principle

Negative Reaction

3’

G G G G T G C C C T A A A A

G T T T T

G G G G A C C C C A C G G G A T T T T

A GC

5’ 3’

5’

5’

XX

XX

Negative Positive BlankPositive

PCR-SSP Data Interpretation

Micro SSP Class I GenericPrimer Set Tray

96 wells/testSame procedure for both Class I and Class IIAllows low resolution typing of Class I allelesA11/23,B49/52, Cw07/12

SSOSequence Specific Oligonucleotides Probe

Hybridisation of amplified PCR products of sample DNA to sequence specific oligonucleotides

Assay can be performed manually or automated

Method

4 – Step Process

DNA Extraction

Any high quality DNA purification system

can be used - but need high quality DNA 200ng of purified DNA required in a

volume of 15µl PCR Amplification

Strip Hybridyzation

Results Interpretation

Dynal RELI™ SSO

Nylon Membrane

Biotin

Streptavidin

Horseradish Peroxidase

PCRProduct

TMB

TMB

Linker = BSA

Probe

Target

H2O2

Method OverviewASSAY CAN BE PERFORMED MANUALLY IN WATERBATH, Baby Bee OR

AUTOMATED USING AutoRELI Mk II

Aspirate & add washsolution

Add PCR amplicon and hybridisation fluid

Hybridize @ 50C for 30min

Aspirate & add wash solution

Wash @ room temp

Aspirate & add substrates A & B

Interpret Results

Shake @ room temp for 10 min

Wash @ room temp for 15 min

Stringent wash @ 50C

Aspirate and add SA-HRP

Wash 5 min @ room temp x2

SBT Method

SBT (Sequencing Base Typing)

1. DNA Extraction

2. Group-specific PCR

3. Sequencing Reaction

4. Electrophoresis/Fluorescence Detection

5. Sequence Analysis

CharacteristicsGold Standard

Labor Intensive

Low Throughput

Capital Cost

ddA

ddC

ddG

ddT A C G T

CGAT G/T GGATC A/G TTCA

CGAT G GGATC A TTCACGAT G GGATC G TTCACGAT T GGATC A TTCACGAT T GGATC G TTCA

Application for HLA Typing

Matching suitable donor for recipients awaiting transplantDisease association testingPaternity testingVaccine efficacy studyDisease resistance study

SEROLOGY SPLIT DNA B40 B60 B*4001/07/10/31/34

B61 B*4002~04/06/09/16/27/29B40 B*4011

B4005 B*4005B21 B*4026

Unknown Other B*40 alleles

SEROLOGY SPLIT DNA

B15 B63 B*1516/17B70 B*1509/37/51B71 B*1510/18B72 B*1503/46B75 B*1502/08/11/21/31B76 B*1512/14/19B77 B*1513

B15 Unknown B*1528~29/33~34/55/58BLANK B*1501102N

B*1526NB35 B*1522Unknown Other B*15 alleles