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Laboratory orientation
Ping Chin Lai
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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.
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T-Cell
Variable Region
Constant Region
Antigen Peptide
MHC
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Peptide Binding Groove
MHC II MHC I
Human Leukocyte Antigens (HLA)
2M
All Nucleated CellsB-Cells, Macrophages
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HLA Typing in Laboratory
Microlymphocytoxicity assays
HLA Class I
Mixed lymphocyte culture
HLA Class II
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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
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DNA based Typing Methods
SSP
RFLPrev. SSOP
SSOP
SBT
PCR
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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
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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’
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230
Sequence-Specific Amplification
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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
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Negative Positive BlankPositive
PCR-SSP Data Interpretation
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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
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SSOSequence Specific Oligonucleotides Probe
Hybridisation of amplified PCR products of sample DNA to sequence specific oligonucleotides
Assay can be performed manually or automated
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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
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Dynal RELI™ SSO
Nylon Membrane
Biotin
Streptavidin
Horseradish Peroxidase
PCRProduct
TMB
TMB
Linker = BSA
Probe
Target
H2O2
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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
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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
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CGAT G/T GGATC A/G TTCA
CGAT G GGATC A TTCACGAT G GGATC G TTCACGAT T GGATC A TTCACGAT T GGATC G TTCA
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Application for HLA Typing
Matching suitable donor for recipients awaiting transplantDisease association testingPaternity testingVaccine efficacy studyDisease resistance study
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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
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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
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