1. Laboratory orientation
Ping Chin Lai

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

3. Antigen Peptide
T-Cell
Constant Region
Variable Region
MHC

5. Peptide Binding Groove
Human Leukocyte Antigens (HLA)
MHC II
MHC I
Peptide Binding Groove    
2M   
B-Cells, Macrophages
All Nucleated Cells

6. HLA Typing in Laboratory
Microlymphocytoxicity assays
HLA Class I
Mixed lymphocyte culture
HLA Class II

7. 原理 Microlymphocytotoxicity test (微量淋巴球細胞毒殺試驗) 已知 未知
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 已知
Eosin/
Flurorescent
Satin
Rabbit
Complement
Read
Cell
Lysis
Serum +
Target
Lymphocytes
Formalin 未知

8. DNA based Typing Methods
RFLP
rev. SSOP
SSOP
SBT
PCR
SSP

9. PCR-SSP Method PCR-SSP (Sequence Specific Primer) Characteristic
1. DNA Extraction
2. PCR using SSP’s
3. Agarose Gel Electrophoresis
Characteristic
Typing Occurs During Amplification
Establish linkages between polymorphisms
Throughput Depends on the # of thermocyclers
Rapid
Matched
Amplification No
Mismatched

10. PCR-SSP Principle Positive Reaction 5’ 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 G C 5’ 3’

11. Sequence-Specific Amplification2 30

12. X PCR-SSP Principle Negative Reaction 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 G C 5’ 3’ X 3’

13. PCR-SSP Data Interpretation
Negative
Positive
Positive
Blank

14. Micro SSP Class I Generic Primer Set Tray
96 wells/test
Same procedure for both Class I and Class II
Allows low resolution typing of Class I alleles
A11/23,B49/52, Cw07/12

15. SSO Sequence Specific Oligonucleotides Probe
Hybridisation of amplified PCR products of sample DNA to sequence specific oligonucleotides
Assay can be performed manually or automated

16. 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

17. Dynal RELI™ SSO TMB Horseradish Peroxidase Streptavidin Target PCR
Nylon Membrane
Biotin
Streptavidin
Horseradish Peroxidase
PCR
Product
TMB
Linker = BSA
Probe
Target
H2O2

18. Method Overview
ASSAY CAN BE PERFORMED MANUALLY IN WATERBATH, Baby Bee OR AUTOMATED USING AutoRELI Mk II
Add PCR amplicon and hybridisation fluid
50C for 30min
Stringent 50C
Aspirate and add SA-HRP
Aspirate & add wash
solution
Wash 5 room temp x2
room temp
Aspirate & add
wash solution
Aspirate & add substrates A & B
room temp for 10 min
room temp for 15 min
Interpret Results

19. SBT Method SBT (Sequencing Base Typing) Characteristics
1. DNA Extraction
2. Group-specific PCR
3. Sequencing Reaction
4. Electrophoresis/Fluorescence Detection
5. Sequence Analysis
Characteristics
Gold Standard
Labor Intensive
Low Throughput
Capital Cost
ddA
ddC
ddG
ddT
A C G T

20. CGAT G/T GGATC A/G TTCA
CGAT G GGATC A TTCA
CGAT G GGATC G TTCA
CGAT T GGATC A TTCA
CGAT T GGATC G TTCA

21. Application for HLA Typing
Matching suitable donor for recipients awaiting transplant
Disease association testing
Paternity testing
Vaccine efficacy study
Disease resistance study

22. SEROLOGY SPLIT DNA
B40 B60 B*4001/07/10/31/34
B61 B*4002~04/06/09/16/27/29
B40 B*4011
B4005 B*4005
B21 B*4026
Unknown Other B*40 alleles

23. SEROLOGY SPLIT DNA
B15 B63 B*1516/17
B70 B*1509/37/51
B71 B*1510/18
B72 B*1503/46
B75 B*1502/08/11/21/31
B76 B*1512/14/19
B77 B*1513
B15 Unknown B*1528~29/33~34/55/58
BLANK B* N
B*1526N
B35 B*1522
Unknown Other B*15 alleles